MicroRNA Expression Abnormalities in Pancreatic Endocrine and Acinar Tumors

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of pancreatic cancer. The invention also provides methods of identifying anti-pancreatic cancer agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 12/700,286filed February 4, 210, now allowed, which claims the benefit of U.S.application Ser. No. 12/160,064, filed Jul. 3, 2008, now U.S. Pat. No.7,670,840 issued Mar. 2, 2010, which claims priority toPCT/US2007/000024, filed Jan. 3, 2007, which is a non-provisionalapplication of Ser. No. 60/756,502 filed Jan. 5, 2006, the entiredisclosures of which are expressly incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by Program ProjectGrants P01CA76259 and P01CA81534 from the National Cancer Institute. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The Sequence Listing, filed electronically and identified as604_(—)51655_SEQ_LIST_OSURF-06139-2, was created on Feb. 9, 2010, is 92kb in size and is hereby incorporated by reference. The electronicallyfiled Sequence Listing is identical to that filed in the U.S. nationalstage application, U.S. Ser. No. 12/160,064 (filed Jul. 3, 2008), ofwhich the instant application is a divisional.

BACKGROUND OF THE INVENTION

Pancreatic cancers can be classified according to where in the pancreasthe cancer is found or according to the type of cell the cancer hasoriginated from. Pancreatic cancer can occur in the head, body or tailof the pancreas and symptoms can vary depending on where in the pancreasthe tumor is located. 70-80% of pancreatic cancers occur in the head ofthe pancreas. The majority of cancers of the pancreas are exocrine intype, and greater than 90% of these exocrine pancreatic cancers areadenocarcinomas. Nearly all of these are ductal adenocarcinomas, whereinthe cancer occurs in the cells lining the ducts of the pancreas. Inaddition, there are rarer types of exocrine pancreatic cancer, such ascystic tumors, cancer of the acinar cells and sarcomas. Cystic tumorsare tumors that cause a cyst or fluid-filled sac in the pancreas.Sarcomas, a cancer of the connective tissue holding together the cellsof the pancreas, are rare and most often occur in children.

In addition to exocrine cancers, endocrine cancers of the pancreas canoccur. The endocrine cancers can be named by reference to the hormonethat they produce, e.g., gastrinomas (which produce gastrin),insulinomas (which produce insulin), somatostatinomas (which producesomatostatin), VIPomas (which produce VIP) and glucagonomas (whichproduce glucagon). In addition, lymphomas of the pancreas can occur,although they are rare.

Pancreatic endocrine tumors (PET) may occur either sporadically or aspart of multiple endocrine neoplasia type 1 (MEN1) syndrome (Kloppel,G., et al., Ann. N.Y. Acad. Sci. 1014:13-27 (2004)). These neoplasms areclinically classified as functioning (F-PET) or nonfunctioning (NF-PET),according to the presence of symptoms due to hormone hypersecretion.F-PETs are mainly represented by insulinomas. At diagnosis, metastaticdisease is observed in only 10% of insulinomas but in up to 60% ofNF-PETs, and most PET-related deaths are caused by liver metastasis(Kloppel, G., et al., Ann. N.Y. Acad. Sci. 1014:13-27 (2004)). Themalignant potential among PETs varies greatly and cannot be predicted onthe basis of histological appearance. In fact, the vast majority of PETsare well-differentiated endocrine tumors (WDET) and are defined aswell-differentiated endocrine carcinomas (WDEC) only when invasion ormetastases are identified (Kloppel, G., et al., Ann. N.Y. Acad. Sci.1014:13-27 (2004)).

Pancreatic acinar cell carcinoma (PACC) is an extremely rare tumor typedistinct from ductal adenocarcinoma and PET, although some overlap withPET is observed by both the expression of neuroendocrine markers in onethird of the cases and the existence of mixed acinar-endocrinecarcinomas (Ohike, N., et al., Virchows Arch. 445:231-35 (2004)). PACCis always malignant with a median survival of 18 months, which liesbetween that of pancreatic ductal adenocarcinoma and endocrine neoplasms(6 months and 40 months, respectively) (Holen, K. D., et al., J. Clin.Oncol. 20:4673-78 (2002)).

Little is known about the molecular pathogenesis of PETs (Kloppel, G.,et al., Ann. N.Y. Acad. Sci. 1014:13-27 (2004)). Inactivation of MEN1gene is the most frequent genetic event identified in sporadic PET,while mutations in genes typically involved in pancreatic adenocarcinomaare uncommon (Perren, A., et al., Ann. N.Y. Acad. Sci. 1014:199-208(2004)). Even less is known regarding the molecular anomalies of PACC(Abraham, S. C., et al., Am. J. Pathol. 160:953-62 (2002)). No geneexpression profile data is available for PACC and our understanding ofgene expression changes that occur in PET is still at an initial phase(Hansel, D. E., et al., Clin. Cancer Res. 10:6152-58 (2004)).

MicroRNAs are small (20-24 nucleotides) noncoding RNA gene products thatserve critical roles in many biological processes, such as cellproliferation, apoptosis and developmental timing. To perform thesefunctions, microRNAs negatively regulate the stability and/ortranslational efficiency of their target mRNAs (Ambros, V., Nature431:350-55 (2004)). Currently, 313 unique mature human microRNAs areknown, 223 of which have been experimentally verified in humans(www.microrna.sanger.ac.uk). Recent studies suggest that aberrantexpression of particular miRNAs may be involved in human diseases, suchas neurological disorders (Ishizuka, A., et al., Genes Dev. 16:2497-2508(2002)) and cancer. In particular, misexpression of miR-16-1 and/ormiR-15a has been found in human chronic lymphocytic leukemias (CLL)(Calin, G. A., et al., Proc. Natl. Acad. Sci. U.S.A. 99:15524-15529(2002)). Aberrant expression of microRNAs has been linked to cancers anddiagnostic/prognostic characteristics of specific cancer types can bedistinguished based on their microRNA profiles (Caldas, C., and J. D.Brenton, Nature Med. 11:712-14 (2005); Croce, C. M., and G. A. Calin,Cell 122:6-7 (2005)). Functional studies also have linked aberrantmicroRNA expression to carcinogenesis (Chan, J. A., et al., Cancer Res.65:6029-33 (2005); Cheng, A. M., et al., Nucleic Acids Res. 33:1290-97(2005); He, L., et al., Nature 435:828-33 (2005); and Johnson, S. M., etal., Cell 120:635-47 (2005)).

The development and use of microarrays containing all known humanmicroRNAs has permitted a simultaneous analysis of the expression ofevery miRNA in a sample (Liu, C. G., et al., Proc Natl. Acad. Sci.U.S.A. 101:9740-9744 (2004)). These microRNA microarrays have not onlybeen used to confirm that miR-16-1 is deregulated in human CLL cells,but also to generate miRNA expression signatures that are associatedwith well-defined clinicopathological features of human CLL (Calin, G.A., et al., Proc. Natl. Acad. Sci. U.S.A. 101:1175-11760 (2004)).

Identification of microRNAs that are differentially-expressed inpancreatic cancer cells may help pinpoint specific miRNAs that areinvolved in pancreatic cancer (e.g., pancreatic endocrine tumors, acinarcarcinomas). Furthermore, the identification of putative targets ofthese miRNAs may help to unravel their pathogenic role. The presentinvention provides novel methods and compositions for the diagnosis,prognosis and treatment of pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofspecific miRNAs associated with altered expression levels in pancreaticcancer cells.

Accordingly, the invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, pancreatic cancer. Accordingto the methods of the invention, the level of at least one miR geneproduct in a test sample from the subject is compared to the level of acorresponding miR gene product in a control sample. An alteration (e.g.,an increase, a decrease) in the level of the miR gene product in thetest sample, relative to the level of a corresponding miR gene productin the control sample, is indicative of the subject either having, orbeing at risk for developing, pancreatic cancer.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample. In another embodiment, the at least onemiR gene product is selected from the group consisting of miR-103-2,miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a,miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2,miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25,miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b,miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a,miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2,miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v,miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3,miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375 and a combination thereof. In yet anotherembodiment, the at least one miR gene product is selected from the groupconsisting of miR-103, miR-107 and a combination thereof. In stillanother embodiment, the at least one miR gene product is selected fromthe group consisting of miR-23a, miR-26b, miR-192, miR-342 and acombination thereof.

In one embodiment, the level of the at least one miR gene product in thetest sample is less than the level of the corresponding miR gene productin the control sample. In another embodiment, the at least one miR geneproduct is selected from the group consisting of miR-326, miR-155,miR-339, miR-34c, miR-345, miR-152, miR-372, miR-128a and a combinationthereof. In yet another embodiment, the at least one miR gene product ismiR-155.

In one embodiment, the at least one miR gene product is selected fromthe group consisting of miR-103, is miR-107, miR-155 and a combinationthereof. In another embodiment, the at least one miR gene product ismiR-103, which is upregulated in the test sample, as compared to thecontrol sample. In yet another embodiment, the at least one miR geneproduct is miR-107, which is upregulated in the test sample, as comparedto the control sample. In still another embodiment, the at least one miRgene product is miR-155, which is downregulated in the test sample, ascompared to the control sample. In a particular embodiment, all three ofthese miRs (miR-103, miR-107 and miR-155) are compared to thecorresponding miRs in the control sample.

In one embodiment, the pancreatic cancer that is diagnosed is apancreatic endocrine tumor (PET). In another embodiment, the pancreaticcancer that is diagnosed is a pancreatic exocrine tumor (e.g., anadenocarcinoma). In yet another embodiment, the pancreatic cancer thatis diagnosed is selected from the group consisting of a pancreaticendocrine tumor (PET) and a pancreatic exocrine tumor (e.g., anadenocarcinoma). In a particular embodiment, the pancreatic cancer thatis diagnosed is selected from the group consisting of an acinar cellcarcinoma (PACC) and an insulinoma. In yet another embodiment, thepancreatic cancer that is diagnosed is selected from the groupconsisting of a pancreatic endocrine tumor (PET), a pancreatic acinarcell carcinoma (PACC) and an insulinoma. In still another embodiment,the diagnostic method can be used to diagnose any type of pancreaticcancer.

In one embodiment, the invention is a method of diagnosing whether asubject has, or is at risk for developing, pancreatic acinar cellcarcinoma (PACC). In this method, the level of at least one miR geneproduct in a test sample from the subject is compared to the level of acorresponding miR gene product in a control sample. An alteration (e.g.,an increase, a decrease) in the level of the miR gene product in thetest sample, relative to the level of a corresponding miR gene productin a control sample, is indicative of the subject either having, orbeing at risk for developing, PACC. In one embodiment, the level of theat least one miR gene product in the test sample is greater than thelevel of the corresponding miR gene product in the control sample. Inanother embodiment, the at least one miR gene product is selected fromthe group consisting of miR-103-2, miR-25, miR-200c, miR-335, miR-21,miR-103-1, miR-92-1, miR-181b-2, miR-191, miR-93, miR-26a-1, miR-17,miR-20, miR-107, miR-26b, miR-215, miR-92-2, miR-192, miR-342, miR-100,miR-3p21-v, miR-106a, miR-15a, miR-23a, miR-181b-1, miR-128b, miR-106b,miR-194-1, miR-219-1, miR-242 and a combination thereof. In yet anotherembodiment, the level of the at least one miR gene product in the testsample is less than the level of the corresponding miR gene product inthe control sample. In still another embodiment, the at least one miRgene product is selected from the group consisting of miR-218-2,miR-339, miR-326, miR-34c, miR-152, miR-138-2, miR-128a and acombination thereof.

In one embodiment, the invention is a method of diagnosing the type ofpancreatic cancer that a subject has. In this method, the level of atleast one miR gene product in a test sample from the subject is comparedto the level of a corresponding miR gene product in a control sample. Analteration (e.g., an increase, a decrease) in the level of the miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of the type ofpancreatic cancer.

In one embodiment, the type of pancreatic cancer that is diagnosed isselected from the group consisting of a pancreatic endocrine tumor (PET)and a pancreatic acinar cell carcinoma (PACC). In another embodiment,the level of the at least one miR gene product in the test sample isgreater than the level of the corresponding miR gene product in thecontrol sample. In another embodiment, the type of pancreatic cancer isa pancreatic endocrine tumor (PET) and the at least one miR gene productis selected from the group consisting of miR-125a, miR-99a, miR-99b,miR-125b-1, miR-342, miR-130a, miR-100, miR-132, miR-129-2, miR-125b-2and a combination thereof. In yet another embodiment, the level of theat least one miR gene product in the test sample is less than the levelof the corresponding miR gene product in the control sample. In stillanother embodiment, the type of pancreatic cancer is a pancreatic acinarcell carcinoma (PACC) and the at least one miR gene product is selectedfrom the group consisting of miR-125a, miR-99a, miR-99b, miR-125b-1,miR-342, miR-130a, miR-100, miR-132, miR-129-2, miR-125b-2 and acombination thereof.

In one embodiment, the type of pancreatic cancer that is diagnosed isselected from the group consisting of a well-differentiated endocrinecarcinoma (WDEC) and a pancreatic acinar cell carcinoma (PACC). Inanother embodiment, the level of the at least one miR gene product inthe test sample is greater than the level of the corresponding miR geneproduct in the control sample. In yet another embodiment, the type ofpancreatic cancer is a well-differentiated endocrine carcinoma (WDEC)and the at least one miR gene product is selected from the groupconsisting of miR-125a, miR-99a, miR-132 and a combination thereof. Inanother embodiment, the level of the at least one miR gene product inthe test sample is less than the level of the corresponding miR geneproduct in the control sample. In still another embodiment, the type ofpancreatic cancer is a well-differentiated endocrine carcinoma (WDEC)and the at least one miR gene product is miR-148a.

In one embodiment, the type of pancreatic cancer that is diagnosed isselected from the group consisting of an insulinoma and anon-functioning pancreatic endocrine tumor (NF-PET). In one embodiment,the level of the at least one miR gene product in the test sample isgreater than the level of the corresponding miR gene product in thecontrol sample. In another embodiment, the type of pancreatic cancer isan insulinoma and the at least one miR gene product is selected from thegroup consisting of miR-204, miR-203, miR-211 and a combination thereof.

In one embodiment, the invention is a method of determining theprognosis of a subject with pancreatic cancer. In this method, the levelof at least one miR gene product, which is associated with an adverseprognosis in pancreatic cancer, is measured in a test sample (e.g., apancreatic cancer sample) from the subject. An alteration (e.g., anincrease, a decrease) in the level of the miR gene product in the testsample, relative to the level of a corresponding miR gene product in acontrol sample, is indicative of an adverse prognosis. In oneembodiment, the level of the at least one miR gene product in the testsample is greater than the level of the corresponding miR gene productin a control sample. In another embodiment, the at least one miR geneproduct that is measured is miR-21. In yet another embodiment, thepancreatic cancer is associated with metastasis and/or a highproliferation index.

In one embodiment, the invention is a method of determining whether apancreatic cancer in a subject is metastatic. In this method, the levelof at least one miR gene product is measured in a test sample (e.g., apancreatic cancer sample) from the subject. An alteration (e.g., anincrease, a decrease) in the level of the miR gene product in the testsample, relative to the level of a corresponding miR gene product in acontrol sample, is indicative of metastasis. In one embodiment, thelevel of the at least one miR gene product in the test sample is greaterthan the level of the corresponding miR gene product in the controlsample. In another embodiment, the at least one miR gene product ismiR-21.

In one embodiment, the invention is a method of determining whether apancreatic cancer in a subject has a high proliferation index. In thismethod, the level of at least one miR gene product is measured in a testsample (e.g., a pancreatic cancer sample) from the subject. Analteration (e.g., an increase, a decrease) in the level of the miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of a high proliferationindex. In one embodiment, the level of the at least one miR gene productin the test sample is greater than the level of the corresponding miRgene product in the control sample. In another embodiment, the at leastone miR gene product is miR-21.

In one embodiment, the invention is a method of determining theprognosis of a subject with pancreatic cancer. In this method, the levelof PDCD4 is measured in a test sample (e.g., a pancreatic cancer sample)from the subject. An alteration (e.g., an increase, a decrease) in thelevel of PDCD4 in the test sample, relative to the level of PDCD4 in acontrol sample, is indicative of an adverse prognosis. In oneembodiment, the level of PDCD4 in the test sample is less than the levelof PDCD4 in the control sample. In another embodiment, the pancreaticcancer is associated with metastasis and/or a high proliferation index.

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art(e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection). In a particular embodiment, the levelof at least one miR gene product is measured by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides toone or more miRNA-specific probe oligonucleotides (e.g., a microarraythat comprises miRNA-specific probe oligonucleotides) to provide ahybridization profile for the test sample, and comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample. An alteration in the signal of at least one miRNA in thetest sample relative to the control sample is indicative of the subjecteither having, or being at risk for developing, pancreatic cancer. Inone embodiment, the signal of at least one miRNA is upregulated,relative to the signal generated from the control sample. In anotherembodiment, the signal of at least one miRNA is downregulated, relativeto the signal generated from the control sample. In a particularembodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina further embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-103-2, miR-107, miR-103-1, miR-342, miR-100, miR-24-2,miR-23a, miR-125a, miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368,miR-26b, miR-125b-2, miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2,miR-21, miR-25, miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17,miR-99b, miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197,miR-10a, miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1,miR-29b-2, miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223,miR-3p21-v, miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214,miR-7-3, miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375, miR-326, miR-155, miR-339, miR-34c, miR-345,miR-152, miR-372, miR-128a and a combination thereof.

The invention also provides methods of diagnosing whether a subject has,or is at risk for developing, a pancreatic cancer with an adverseprognosis. In this method, the level of at least one miR gene product,which is associated with an adverse prognosis in pancreatic cancer, ismeasured by reverse transcribing RNA from a test sample obtained fromthe subject to provide a set of target oligodeoxynucleotides. The targetoligodeoxynucleotides are then hybridized to one or more miRNA-specificprobe oligonucleotides (e.g., a microarray that comprises miRNA-specificprobe oligonucleotides) to provide a hybridization profile for the testsample, and the test sample hybridization profile is compared to ahybridization profile generated from a control sample. An alteration inthe signal of at least one miRNA in the test sample relative to thecontrol sample is indicative of the subject either having, or being atrisk for developing, a pancreatic cancer with an adverse prognosis. Inone embodiment, an alteration in the signal of miR-21 is indicative ofthe subject either having, or being at risk for developing, a pancreaticcancer with an adverse prognosis.

The invention also encompasses methods of treating pancreatic cancer ina subject, wherein at least one miR gene product is deregulated (e.g.,downregulated, upregulated) in the cancer cells of the subject. When atleast one isolated miR gene product is downregulated in the pancreaticcancer cells, the method comprises administering an effective amount ofan isolated miR gene product, or an isolated variant orbiologically-active fragment thereof, such that proliferation of cancercells in the subject is inhibited. In one embodiment, the at least oneisolated miR gene product that is administered to the subject isselected from the group consisting of miR-326, miR-155, miR-339,miR-34c, miR-345, miR-152, miR-372, miR-128a and a combination thereof(or an isolated variant or biologically-active fragment of one or moreof these miRs). When at least one isolated miR gene product isupregulated in the cancer cells, the method comprises administering tothe subject an effective amount of at least one compound for inhibitingexpression of the at least one miR gene product, such that proliferationof pancreatic cancer cells is inhibited. In one embodiment, the compoundfor inhibiting expression of the at least one miR gene product inhibitsa miR gene product selected from the group consisting of miR-103-2,miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a,miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2,miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25,miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b,miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a,miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2,miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v,miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3,miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375 and a combination thereof.

In a related embodiment, the methods of treating pancreatic cancer in asubject additionally comprise the step of first determining the amountof at least one miR gene product in pancreatic cancer cells from thesubject, and comparing that level of the miR gene product to the levelof a corresponding miR gene product in control cells. If expression ofthe miR gene product is deregulated (e.g., downregulated, upregulated)in pancreatic cancer cells, the methods further comprise altering theamount of the at least one miR gene product expressed in the pancreaticcancer cells. In one embodiment, the amount of the miR gene productexpressed in the cancer cells is less than the amount of the miR geneproduct expressed in control cells, and an effective amount of the miRgene product, or an isolated variant or biologically-active fragmentthereof, is administered to the subject. In another embodiment, theamount of the miR gene product expressed in the cancer cells is greaterthan the amount of the miR gene product expressed in control cells, andan effective amount of at least one compound for inhibiting expressionof the at least one miR gene is administered to the subject. SuitablemiRs and compounds that inhibit expression of miR genes include, forexample, those described herein.

The invention further provides pharmaceutical compositions for treatingpancreatic cancer. In one embodiment, the pharmaceutical compositionscomprise at least one isolated miR gene product, or an isolated variantor biologically-active fragment thereof, and apharmaceutically-acceptable carrier. In a particular embodiment, the atleast one miR gene product corresponds to a miR gene product that has adecreased level of expression in pancreatic cancer cells relative tosuitable control cells (i.e., it is downregulated). In a certainembodiment, the isolated miR gene product is selected from the groupconsisting of miR-326, miR-155, miR-339, miR-34c, miR-345, miR-152,miR-372, miR-128a and a combination thereof.

In another embodiment, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound and apharmaceutically-acceptable carrier. In a particular embodiment, the atleast one miR expression-inhibition compound is specific for a miR geneproduct whose expression is greater in pancreatic cancer cells thancontrol cells (i.e., it is upregulated). In certain embodiments, the miRexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-103-2, miR-107,miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a, miR-26a-1,miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2, miR-125b-1,miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25, miR-92-2,miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b, miR-181b-1,miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a, miR-224,miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2, miR-150,miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v, miR-128b,miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3, miR-29c,miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20, miR-129-1,miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95, miR-222, miR-30e,miR-129-2, miR-345, miR-143, miR-182, miR-1-1, miR-133a-1, miR-200c,miR-194-1, miR-210, miR-181c, miR-192, miR-220, miR-213, miR-323,miR-375 and a combination thereof.

The invention also encompasses methods of identifying an anti-pancreaticcancer agent, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in pancreatic cancer cells. An increase inthe level of the miR gene product in the cell, relative to a suitablecontrol cell, is indicative of the test agent being an anti-pancreaticcancer agent. In a particular embodiment, the at least one miR geneproduct associated with decreased expression levels in pancreatic cancercells is selected from the group consisting of miR-326, miR-155,miR-339, miR-34c, miR-345, miR-152, miR-372, miR-128a and a combinationthereof.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in pancreatic cancer cells. A decreasein the level of the miR gene product associated with increasedexpression levels in pancreatic cancer in the cell, relative to asuitable control cell, is indicative of the test agent being ananti-pancreatic cancer agent. In a particular embodiment, the at leastone miR gene product associated with increased expression levels inpancreatic cancer cells is selected from the group consisting ofmiR-103-2, miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a,miR-125a, miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b,miR-125b-2, miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2, miR-21,miR-25, miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b,miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a,miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2,miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v,miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3,miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375 and a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A depicts an miRNA expression unsupervised hierarchical clusterview of 12 normal pancreas (Normal) and 44 pancreatic tumors, including22 well-differentiated pancreatic endocrine tumors (WDET), 18well-differentiated pancreatic endocrine carcinomas (WDEC) and 4pancreatic acinar cell carcinomas (ACC) (listed at top). WDET samplesincluded 11 insulinomas (INS) and 1 Non-functioning PET (NF); WDECsamples included 1 INS and 17 NF-PET. The analysis was performed usingthe aggregate values of replicate spots obtained applying median polishalgorithm and selecting the first 200 probes with the higherinterquartile range, which contained the mature microRNA sequences.Notably, PACC samples fell in a unique cluster that was part of thewider cluster including all PETs, while there was no distinctive patternbetween insulinomas and NF-PET. As is depicted, a common microRNAexpression pattern distinguishes pancreatic endocrine and acinar tumorsfrom normal pancreas.

FIG. 1B depicts particular microRNAs that are found to be upregulated inPET versus Normal tissue (upregulated microRNAs are listed in red).

FIG. 1C depicts particular microRNAs that are found to be upregulated inPET versus Normal tissue (upregulated microRNAs are listed in red).

FIG. 1D depicts two microRNAs that are upregulated in insulinoma versusNon-functioning PET ((upregulated microRNAs are listed in blue).

FIG. 1E depicts particular microRNAs that are found to be downregulatedin PET versus Normal tissue (downregulated microRNAs are listed ingreen).

FIG. 2A depicts box-and-whiskers plots showing the expression levels ofmiR-103 and miR-155, which were measured by microarray analysis of 12normal pancreas (Normal) and 44 pancreatic tumors, including 22well-differentiated pancreatic endocrine tumors (WDET), 18well-differentiated pancreatic endocrine carcinomas (WDEC) and 4pancreatic acinar cell carcinomas (ACC). The median intensity ishighlighted by bold lines. As shown, the overexpression of miR-103 andlack of expression of miR-155 is particular to pancreatic insular andacinar tumors.

FIG. 2B depicts Northern blot analysis, which parallels the microarrayexpression data shown in FIG. 2A. 5S rRNA (5S-RNA) served as a loadingcontrol.

FIG. 3A depicts a box-and-whiskers plot showing the expression level ofmiR-204, which was measured by microarray analysis of 12 normal pancreas(Normal), 12 insulinomas, 28 non functioning pancreatic endocrine tumors(NF-PET) and 4 pancreatic acinar cell carcinomas (ACC). The medianintensity is highlighted by bold lines.

FIG. 3B is a graph showing a strong correlation between miR-204expression and insulin staining assessed by immunohistochemistry (IHC).

FIG. 3C depicts Northern blot analysis, which confirms the microarrayexpression data and shows that miR-204 over-expression is specific toinsulinomas. 5S rRNA (5S-RNA) served as a loading control.

FIG. 4A depicts a box-and-whiskers plot showing the different expressionlevel of miR-21, which was measured by microarray analysis, betweenpancreatic endocrine tumors with (Meta+) or without (Meta−) livermetastasis. As is shown, expression of miR-21 is strongly associatedwith the presence of liver metastasis.

FIG. 4B depicts a box-and-whiskers plot showing the different expressionlevel of miR-21, which was measured by microarray analysis, betweentumors with a proliferation index >2% (High) or ≦2% (Low), as measuredby Ki67 immunohistochemistry. As is shown, expression of miR-21 isstrongly associated with tumoral proliferation index.

FIG. 4C depicts Northern blot analysis, which confirms the microarrayexpression data. 5S rRNA (5S-RNA) served as a loading control.

FIG. 5 is a plot showing the expression of miR-21 and PDCD4 mRNA innormal pancreas (*), metastatic (▴) and nonmetastatic (Δ) PET. A Robustlocally weighted regression function has been used to fit a line amongthe data points. As is shown, there is an inverse correlation betweenthe expression of miR-21 and its putative mRNA target PDCD4.

FIG. 6A depicts Northern blot analysis showing overexpression of miR-26band miR-107 in all the pancreatic insulinomas and non functioningendocrine tumors (NF-PET) that were tested. These results validate themicroarray data for the overexpressed microRNAs. 5S rRNA (5S-RNA) servedas a loading control.

FIG. 6B depicts Northern blot analysis showing overexpression of miR-23aand miR-342 in all the pancreatic insulinomas and non functioningendocrine tumors (NF-PET) that were tested. These results validate themicroarray data for the overexpressed microRNAs. 5S rRNA (5S-RNA) servedas a loading control.

FIG. 6C depicts Northern blot analysis showing overexpression of miR-192in four of eight well-differentiated endocrine tumors (WDET), in allfour well-differentiated endocrine carcinomas (WDEC), and one acinarcell carcinoma (ACC). These results validate the microarray data for theoverexpressed microRNA. 5S rRNA (5S-RNA) served as a loading control.

FIG. 7A depicts Northern blot analysis showing that miR-375 is apancreas-specific miR. 5S rRNA (5S-RNA) served as a loading control.

FIG. 7B depicts Northern blot analysis showing that the expression ofmiR-375 is a feature of pancreatic endocrine and acinar tumors,irrespective of the presence (insulinomas) or absence (NF-PET) ofclinically evident insulin oversecretion. NF-PET, nonfunctioningpancreatic endocrine tumors. 5S rRNA (5S-RNA) served as a loadingcontrol. As is shown, mir-375 expression is common in pancreatic insularand acinar tumors.

FIG. 7C depicts Northern blot analysis showing that the expression ofmiR-375 is a feature of pancreatic endocrine and acinar tumors,irrespective of the presence (insulinomas) or absence (NF-PET and ACC)of clinically evident insulin oversecretion. NF-PET, nonfunctioningpancreatic endocrine tumors; ACC, pancreatic acinar cell carcinomas. 5SrRNA (5S-RNA) served as a loading control. As is shown, mir-375expression is common in pancreatic insular and acinar tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the identification ofparticular microRNAs having altered expression in pancreatic cancercells relative to normal control cells, and on association of thesemicroRNAs with particular diagnostic, prognostic and therapeuticfeatures. As described herein:

a common pattern of microRNA expression distinguishes pancreatic tumortypes from normal pancreas, and thereby implicates the involvement ofparticular microRNAs in pancreatic tumorigenesis;

the expression of miR-103 and miR-107, associated with lack ofexpression of miR-155, discriminates pancreatic tumors from normalpancreas;

at least 10 microRNAs distinguishes endocrine tumors from acinar tumors,and implicates particular microRNAs in endocrine differentiation and/orendocrine tumorigenesis;

miR-204 is primarily expressed in insulinomas and correlates withimmunohistochemical expression of insulin; and

over-expression of miR-21 is strongly associated with both a high Ki67proliferation index and the presence of liver metastasis.

These results imply that alteration in microRNA expression is related toendocrine and acinar neoplastic transformation and progression ofmalignancy. Accordingly, expression of particular microRNAs, as well asalterations of such microRNA expression, can be used in the diagnostic,prognostic and therapeutic methods described herein.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed or processed RNA transcript from amiR gene. As the miR gene products are not translated into protein, theterm “miR gene products” does not include proteins. The unprocessed miRgene transcript is also called a “miR precursor,” and typicallycomprises an RNA transcript of about 70-100 nucleotides in length. ThemiR precursor can be processed by digestion with an RNAse (for example,Dicer, Argonaut, RNAse III (e.g., E. coli RNAse III)) into an active19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA moleculeis also called the “processed” miR gene transcript or “mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without having tobe processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

The present invention encompasses methods of diagnosing whether asubject has, or is at risk for developing, pancreatic cancer, comprisingmeasuring the level of at least one miR gene product in a test samplefrom the subject and comparing the level of the miR gene product in thetest sample to the level of a corresponding miR gene product in acontrol sample. As used herein, a “subject” can be any mammal that has,or is suspected of having, pancreatic cancer. In a preferred embodiment,the subject is a human who has, or is suspected of having, pancreaticcancer.

The pancreatic cancer can be any form of pancreatic cancer, for example,pancreatic cancers of differing histology (e.g., exocrine tumors,endocrine tumors, carcinomas, lymphomas). In one embodiment, thepancreatic cancer that is diagnosed is a pancreatic endocrine tumor(PET). In another embodiment, the pancreatic cancer that is diagnosed isa pancreatic exocrine tumor (e.g., an adenocarcinoma). In yet anotherembodiment, the pancreatic cancer that is diagnosed is selected from thegroup consisting of a pancreatic endocrine tumor (PET) and a pancreaticexocrine tumor (e.g., an adenocarcinoma). In a particular embodiment,the pancreatic cancer that is diagnosed is selected from the groupconsisting of an acinar cell carcinoma (PACC) and an insulinoma. In yetanother embodiment, the pancreatic cancer that is diagnosed is selectedfrom the group consisting of a pancreatic endocrine tumor (PET), apancreatic acinar cell carcinoma (PACC) and an insulinoma. Furthermore,as described herein, the pancreatic cancer may be associated with aparticular prognosis (e.g., low survival rate, fast progression).

Tables 1a and 1b depict the nucleotide sequences of particular precursorand mature human microRNAs.

TABLE 1a  Human microRNA Precursor Sequences. Precursor  SEQ ID NameSequence (5′ To 3′)* NO. let-7a-1CACUGUGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACAC 1CCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUUCCUAACG  UG let-7a-2AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAAGGGA 2GAUAACUGUACAGCCUCCUAGCUUUCCU let-7a-3GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAU 3GGGAUAACUAUACAAUCUACUGUCUUUCCU let-7a-4GUGACUGCAUGCUCCCAGGUUGAGGUAGUAGGUUGUAUAGUUUA 4GAAUUACACAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCUU  GGGUCUUGCACUAAACAAC let-7bGGCGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAGUGAUGU 5UGCCCCUCGGAAGAUAACUAUACAACCUACUGCCUUCCCUG let-7cGCAUCCGGGUUGAGGUAGUAGGUUGUAUGGUUUAGAGUUACACC 6CUGGGAGUUAACUGUACAACCUUCUAGCUUUCCUUGGAGC let-7dCCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAGGGAUUU 7UGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG let-7d-v1 CUAGGAAGAGGUAGUAGUUUGCAUAGUUUUAGGGCAAAGAUUUU 8GCCCACAAGUAGUUAGCUAUACGACCUGCAGCCUUUUGUAG let-7d-v2 CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGUGACAUU 9GCCCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUAG let-7eCCCGGGCUGAGGUAGGAGGUUGUAUAGUUGAGGAGGACACCCAA 10GGAGAUCACUAUACGGCCUCCUAGCUUUCCCCAGG let-7f-1UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGUGAUUUU 11ACCCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUGA let-7f-2-1 CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGUGAUU 12UUACCCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUG A let-7f-2-2 CUGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUAGGGUCAUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUUUCCCACGG 13 let-7gUUGCCUGAUUCCAGGCUGAGGUAGUAGUUUGUACAGUUUGAGGGUCUAUGAUACCACCCGGUACAGGAGAUAACUGUACAGGCCACUG 14 CCUUGCCAGGAACAGCGCGClet-7i CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGUGACAUU 15GCCCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUAG miR-1b-1-1ACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGAACAUA 16CAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGC miR-1b-1-2CAGCUAACAACUUAGUAAUACCUACUCAGAGUACAUACUUCUUU 17AUGUACCCAUAUGAACAUACAAUGCUAUGGAAUGUAAAGAAGUA UGUAUUUUUGGUAGGCAAUAmiR-1b-2 GCCUGCUUGGGAAACAUACUUCUUUAUAUGCCCAUAUGGACCUG 18CUAAGCUAUGGAAUGUAAAGAAGUAUGUAUCUCAGGCCGGG miR-1bUGGGAAACAUACUUCUUUAUAUGCCCAUAUGGACCUGCUAAGCU 19AUGGAAUGUAAAGAAGUAUGUAUCUCA miR-1dACCUACUCAGAGUACAUACUUCUUUAUGUACCCAUAUGAACAUA 20CAAUGCUAUGGAAUGUAAAGAAGUAUGUAUUUUUGGUAGGC miR-7-1aUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUG 21UUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUG GCACAGGCCAUGCCUCUACAmiR-7-1b UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUU 22GUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAU GGCACAGGCCAUGCCUCUACAGmiR-7-2 CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGACUAGUG 23AUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUA CCUAAUGGUGCCAGCCAUCGCAmiR-7-3 AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGACUAGUGA 24UUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCU CAUAGCGCAGACUCCCUUCGACmiR-9-1 CGGGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGGUG 25UGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUAACCCC A miR-9-2GGAAGCGAGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGUAU 26UGGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAACUCCUUCA miR-9-3GGAGGCCCGUUUCUCUCUUUGGUUAUCUAGCUGUAUGAGUGCCA 27CAGAGCCGUCAUAAAGCUAGAUAACCGAAAGUAGAAAUGAUUCU CA miR-10aGAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAAUUUGUG 28UAAGGAAUUUUGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUA GUUGACAUAAACACUCCGCUCUmiR-10b CCAGAGGUUGUAACGUUGUCUAUAUAUACCCUGUAGAACCGAAU 29UUGUGUGGUAUCCGUAUAGUCACAGAUUCGAUUCUAGGGGAAUA UAUGGUCGAUGCAAAAACUUCAmiR-15a-2 GCGCGAAUGUGUGUUUAAAAAAAAUAAAACCUUGGAGUAAAGUA 30GCAGCACAUAAUGGUUUGUGGAUUUUGAAAAGGUGCAGGCCAUA UUGUGCUGCCUCAAAAAUACmiR-15a CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAA 31AAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG miR-15b-1CUGUAGCAGCACAUCAUGGUUUACAUGCUACAGUCAAGAUGCGA 32 AUCAUUAUUUGCUGCUCUAGmiR-15b-2 UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAUGGUUUACAUGC 33UACAGUCAAGAUGCGAAUCAUUAUUUGCUGCUCUAGAAAUUUAA GGAAAUUCAU miR-16-1GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUC 34UAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGA C miR-16-2GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAAAUAUAU 35AUUAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC miR-16-13GCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUAAA 36AUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGU miR-17GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGU 37GCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC miR-18UGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAGUAGAUUAGCAU 38CUACUGCCCUAAGUGCUCCUUCUGGCA miR-18-13UUUUUGUUCUAAGGUGCAUCUAGUGCAGAUAGUGAAGUAGAUUA 39GCAUCUACUGCCCUAAGUGCUCCUUCUGGCAUAAGAA miR-19aGCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAAGAAUG 40UAGUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUGC miR-19a-13 CAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAAGAAUGU 41AGUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUG miR-19b-1CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUG 42AUAUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUG miR-19b-2ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGU 43AUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGU GAUAAUGU miR-19b-13 UUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAUAUU 44CUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAG miR-19b-XUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAUAUGUAU 45AUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAU miR-20GUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGUUUAGUUAUCUA 46 (miR-20a)CUGCAUUAUGAGCACUUAAAGUACUGC miR-21UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUG 47GCAACACCAGUCGAUGGGCUGUCUGACA miR-21-17ACCUUGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCU 48CAUGGCAACACCAGUCGAUGGGCUGUCUGACAUUUUG miR-22GGCUGAGCCGCAGUAGUUCUUCAGUGGCAAGCUUUAUGUCCUGA 49CCCAGCUAAAGCUGCCAGUUGAAGAACUGUUGCCCUCUGCC miR-23aGGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAA 50AUCACAUUGCCAGGGAUUUCCAACCGACC miR-23bCUCAGGUGCUCUGGCUGCUUGGGUUCCUGGCAUGCUGAUUUGUG 51ACUUAAGAUUAAAAUCACAUUGCCAGGGAUUACCACGCAACCAC GACCUUGGC miR-23-19CCACGGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUC 52ACAAAUCACAUUGCCAGGGAUUUCCAACCGACCCUGA miR-24-1CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACU 53 GGCUCAGUUCAGCAGGAACAGGAGmiR-24-2 CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGGUUUGUG 54UACACUGGCUCAGUUCAGCAGGAACAGGG miR-24-19CCCUGGGCUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGG 55UUUGUGUACACUGGCUCAGUUCAGCAGGAACAGGGG miR-24-9CCCUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACAC 56UGGCUCAGUUCAGCAGGAACAGCAUC miR-25GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUG 57CCCUGGGCAUUGCACUUGUCUCGGUCUGACAGUGCCGGCC miR-26aAGGCCGUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGU 58CCCAAUGGCCUAUCUUGGUUACUUGCACGGGGACGCGGGCCU miR-26a-1guggccucguucaaguaauccaggauaggcugugcaggucccaaugggccuauucuugguuacuug 59cacggggacgc miR-26a-2ggcuguggcuggauucaaguaauccaggauaggcuguuuccaucugugaggccuauucuugauuac 60uuguuucuggaggcagcu miR-26b CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCC61 AGCCUGUUCUCCAUUACUUGGCUCGGGGACCGG miR-27aCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCACACCAA 62GUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAG miR-27b-1 AGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAUUGGUUUCCGC 63UUUGUUCACAGUGGCUAAGUUCUGCACCU miR-27b-2 ACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUG 64AUUGGUUUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAA GAGAAGGUG miR-27-19 CCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCACACCA 65AGUCGUGUUCACAGUGGCUAAGUUCCGCCCCCCAGG miR-28GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUGAGUUACCUUUC 66UGACUUUCCCACUAGAUUGUGAGCUCCUGGAGGGCAGGCACU miR-29a-2 CCUUCUGUGACCCCUUAGAGGAUGACUGAUUUCUUUUGGUGUUC 67AGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAUAAU GAUUGGGGAAGAGCACCAUGmiR-29a AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAG 68CACCAUCUGAAAUCGGUUAU miR-29b-1 cuucaggaagcugguuucauauggugguuuagauuuaaauagugauugucuagcaccauuugaaau 69caguguucuuggggg miR-29b-2 cuucuggaagcugguuucacaugguggcuuagauuuuuccaucuuuguaucuagcaccauuugaaa 70ucaguguuuuaggag miR-29c ACCACUGGCCCAUCUCUUACACAGGCUGACCGAUUUCUCCUGGUG 71UUCAGAGUCUGUUUUUGUCUAGCACCAUUUGAAAUCGGUUAUGA UGUAGGGGGAAAAGCAGCAGCmiR-30a GCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCACAGAUG 72GGCUUUCAGUCGGAUGUUUGCAGCUGC miR-30b-1 AUGUAAACAUCCUACACUCAGCUGUAAUACAUGGAUUGGCUGGG 73 AGGUGGAUGUUUACGUmiR-30b-2  ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUGUAAUA 74CAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCAGCUGACUUGGA miR-30cAGAUACUGUAAACAUCCUACACUCUCAGCUGUGGAAAGUAAGAA 75AGCUGGGAGAAGGCUGUUUACUCUUUCU miR-30dGUUGUUGUAAACAUCCCCGACUGGAAGCUGUAAGACACAGCUAA 76GCUUUCAGUCAGAUGUUUGCUGCUAC miR-30ecuguaaacauccuugacuggaagcuguaagguguucagaggagcuuucagucggauguuuacag 77miR-31 GGAGAGGAGGCAAGAUGCUGGCAUAGCUGUUGAACUGGGAACCU 78GCUAUGCCAACAUAUUGCCAUCUUUCC miR-32GGAGAUAUUGCACAUUACUAAGUUGCAUGUUGUCACGGCCUCAA 79UGCAAUUUAGUGUGUGUGAUAUUUUC miR-33bGGGGGCCGAGAGAGGCGGGCGGCCCCGCGGUGCAUUGCUGUUGC 80AUUGCACGUGUGUGAGGCGGGUGCAGUGCCUCGGCAGUGCAGCC CGGAGCCGGCCCCUGGCACCACmiR-33b-2  ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUGUAAUA 81CAUGGAUUGGCUGGGAGGUGGAUGUUUACUUCAGCUGACUUGGA miR-33CUGUGGUGCAUUGUAGUUGCAUUGCAUGUUCUGGUGGUACCCAU 82GCAAUGUUUCCACAGUGCAUCACAG miR-34-aGGCCAGCUGUGAGUGUUUCUUUGGCAGUGUCUUAGCUGGUUGUU 83GUGAGCAAUAGUAAGGAAGCAAUCAGCAAGUAUACUGCCCUAGA AGUGCUGCACGUUGUGGGGCCCmiR-34-bgugcucgguuuguaggcagugucauuagcugauuguacuguggugguuacaaucacuaacuccacu 84gccaucaaaacaaggcac miR-34-cagucuaguuacuaggcaguguaguuagcugauugcuaauaguaccaaucacuaaccacacggccagg 85uaaaaagauu miR-91-13 UCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGUG 86CAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGA miR-92-1CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUA 87UGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG miR-92 -2UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUA 88AAGUAUUGCACUUGUCCCGGCCUGUGGAAGA miR-93-1CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCC 89 (miR-93-2)AACCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG miR-95-4AACACAGUGGGCACUCAAUAAAUGUCUGUUGAAUUGAAAUGCGU 90UACAUUCAACGGGUAUUUAUUGAGCACCCACUCUGUG miR-96-7UGGCCGAUUUUGGCACUAGCACAUUUUUGCUUGUGUCUCUCCGC 91UCUGAGCAAUCAUGUGCAGUGCCAAUAUGGGAAA miR-97-6GUGAGCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCACA 92 (miR-30*)GAUGGGCUUUCAGUCGGAUGUUUGCAGCUGCCUACU miR-98GUGAGGUAGUAAGUUGUAUUGUUGUGGGGUAGGGAUAUUAGGCC 93CCAAUUAGAAGAUAACUAUACAACUUACUACUUUCC miR-99bGGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACAC 94AAGCUCGUGUCUGUGGGUCCGUGUC miR-99aCCCAUUGGCAUAAACCCGUAGAUCCGAUCUUGUGGUGAAGUGGA 95CCGCACAAGCUCGCUUCUAUGGGUCUGUGUCAGUGUG miR-100-1/2AAGAGAGAAGAUAUUGAGGCCUGUUGCCACAAACCCGUAGAUCC 96GAACUUGUGGUAUUAGUCCGCACAAGCUUGUAUCUAUAGGUAUG UGUCUGUUAGGCAAUCUCACmiR-100-11 CCUGUUGCCACAAACCCGUAGAUCCGAACUUGUGGUAUUAGUCC 97GCACAAGCUUGUAUCUAUAGGUAUGUGUCUGUUAGG miR-101-1/2AGGCUGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUUC 98UAAAGGUACAGUACUGUGAUAACUGAAGGAUGGCAGCCAUCUUA CCUUCCAUCAGAGGAGCCUCACmiR-101 UCAGUUAUCACAGUGCUGAUGCUGUCCAUUCUAAAGGUACAGUA 99 CUGUGAUAACUGAmiR-101-1 UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUUCUAAA 100GGUACAGUACUGUGAUAACUGAAGGAUGGCA miR-101-2acuguccuuuuucgguuaucaugguaccgaugcuguauaucugaaagguacaguacugugauaacu 101gaagaaugguggu miR-101-9 UGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUGUAUAUCUGAAA 102GGUACAGUACUGUGAUAACUGAAGAAUGGUG miR-102-1CUUCUGGAAGCUGGUUUCACAUGGUGGCUUAGAUUUUUCCAUCU 103UUGUAUCUAGCACCAUUUGAAAUCAGUGUUUUAGGAG miR-102-7.1 CUUCAGGAAGCUGGUUUCAUAUGGUGGUUUAGAUUUAAAUAGUG 104 (miR-102-AUUGUCUAGCACCAUUUGAAAUCAGUGUUCUUGGGGG 7.2) miR-103-2UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUGUAGCAUUCAGG 105UCAAGCAACAUUGUACAGGGCUAUGAAAGAACCA miR-103-1UACUGCCCUCGGCUUCUUUACAGUGCUGCCUUGUUGCAUAUGGA 106UCAAGCAGCAUUGUACAGGGCUAUGAAGGCAUUG miR-104-17AAAUGUCAGACAGCCCAUCGACUGGUGUUGCCAUGAGAUUCAAC 107AGUCAACAUCAGUCUGAUAAGCUACCCGACAAGG miR-105-1UGUGCAUCGUGGUCAAAUGCUCAGACUCCUGUGGUGGCUGCUCA 108UGCACCACGGAUGUUUGAGCAUGUGCUACGGUGUCUA miR-105-2ugugcaucguggucaaaugcucagacuccugugguggcugcuuaugcaccacggauguuugagcau 109gugcuauggugucua miR-106-a CCUUGGCCAUGUAAAAGUGCUUACAGUGCAGGUAGCUUUUUGAG110 AUCUACUGCAAUGUAAGCACUUCUUACAUUACCAUGG miR-106-bccugccggggcuaaagugcugacagugcagauagugguccucuccgugcuaccgcacuguggguac 111uugcugcuccagcagg miR-107 CUCUCUGCUUUCAGCUUCUUUACAGUGUUGCCUUGUGGCAUGGA112 GUUCAAGCAGCAUUGUACAGGGCUAUCAAAGCACAGA miR-108-1-ACACUGCAAGAACAAUAAGGAUUUUUAGGGGCAUUAUGACUGAG 113 smallUCAGAAAACACAGCUGCCCCUGAAAGUCCCUCAUUUUUCUUGCUG U miR-108-2-ACUGCAAGAGCAAUAAGGAUUUUUAGGGGCAUUAUGAUAGUGGA smallAUGGAAACACAUCUGCCCCCAAAAGUCCCUCAUUUU 114 miR-122a-1CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAA 115CUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC miR-122a-2AGCUGUGGAGUGUGACAAUGGUGUUUGUGUCCAAACUAUCAAAC 116 GCCAUUAUCACACUAAAUAGCUmiR-123 ACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAACUCGUAC 117CGUGAGUAAUAAUGCGC miR-124a-1AGGCCUCUCUCUCCGUGUUCACAGCGGACCUUGAUUUAAAUGUCC 118AUACAAUUAAGGCACGCGGUGAAUGCCAAGAAUGGGGCUG miR-124a-2AUCAAGAUUAGAGGCUCUGCUCUCCGUGUUCACAGCGGACCUUG 119AUUUAAUGUCAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGCG GAGCCUACGGCUGCACUUGAAGmiR-124a-3 UGAGGGCCCCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUC 120UAUACAAUUAAGGCACGCGGUGAAUGCCAAGAGAGGCGCCUCC miR-124aCUCUGCGUGUUCACAGCGGACCUUGAUUUAAUGUCUAUACAAUU 121AAGGCACGCGGUGAAUGCCAAGAG miR-124bCUCUCCGUGUUCACAGCGGACCUUGAUUUAAUGUCAUACAAUUA 122 AGGCACGCGGUGAAUGCCAAGAGmiR-125a-1 UGCCAGUCUCUAGGUCCCUGAGACCCUUUAACCUGUGAGGACAUC 123CAGGGUCACAGGUGAGGUUCUUGGGAGCCUGGCGUCUGGCC miR-125a-2GGUCCCUGAGACCCUUUAACCUGUGAGGACAUCCAGGGUCACAG 124 GUGAGGUUCUUGGGAGCCUGGmiR-125b-1 UGCGCUCCUCUCAGUCCCUGAGACCCUAACUUGUGAUGUUUACCG 125UUUAAAUCCACGGGUUAGGCUCUUGGGAGCUGCGAGUCGUGCU miR-125b-2ACCAGACUUUUCCUAGUCCCUGAGACCCUAACUUGUGAGGUAUU 126UUAGUAACAUCACAAGUCAGGCUCUUGGGACCUAGGCGGAGGGG A miR-126-1CGCUGGCGACGGGACAUUAUUACUUUUGGUACGCGCUGUGACAC 127UUCAAACUCGUACCGUGAGUAAUAAUGCGCCGUCCACGGCA miR-126-2ACAUUAUUACUUUUGGUACGCGCUGUGACACUUCAAACUCGUAC 128 CGUGAGUAAUAAUGCGCmiR-127-1 UGUGAUCACUGUCUCCAGCCUGCUGAAGCUCAGAGGGCUCUGAU 129UCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGGUCGGAAGU CUCAUCAUC miR-127-2CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGAUCAUC 130GGAUCCGUCUGAGCUUGGCUGGUCGG miR-128aUGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUAC 131AUUUCUCACAGUGAACCGGUCUCUUUUUCAGCUGCUUC miR-128bGCCCGGCAGCCACUGUGCAGUGGGAAGGGGGGCCGAUACACUGU 132ACGAGAGUGAGUAGCAGGUCUCACAGUGAACCGGUCUCUUUCCC UACUGUGUCACACUCCUAAUGGmiR-128 GUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUACAUUUCU 133CACAGUGAACCGGUCUCUUUUUCAGC miR-129-1UGGAUCUUUUUGCGGUCUGGGCUUGCUGUUCCUCUCAACAGUAG 134UCAGGAAGCCCUUACCCCAAAAAGUAUCUA miR-129-2UGCCCUUCGCGAAUCUUUUUGCGGUCUGGGCUUGCUGUACAUAA 135CUCAAUAGCCGGAAGCCCUUACCCCAAAAAGCAUUUGCGGAGGGC G miR-130aUGCUGCUGGCCAGAGCUCUUUUCACAUUGUGCUACUGUCUGCACC 136UGUCACUAGCAGUGCAAUGUUAAAAGGGCAUUGGCCGUGUAGUG miR-131-1gccaggaggcggGGUUGGUUGUUAUCUUUGGUUAUCUAGCUGUAUGAG 137UGGUGUGGAGUCUUCAUAAAGCUAGAUAACCGAAAGUAAAAAUA ACCCCAUACACUGCGCAGmiR-131-3 CACGGCGCGGCAGCGGCACUGGCUAAGGGAGGCCCGUUUCUCUCU 138UUGGUUAUCUAGCUGUAUGAGUGCCACAGAGCCGUCAUAAAGCU AGAUAACCGAAAGUAGAAAUGmiR-131 GUUGUUAUCUUUGGUUAUCUAGCUGUAUGAGUGUAUUGGUCUUC 139AUAAAGCUAGAUAACCGAAAGUAAAAAC miR-132-1CCGCCCCCGCGUCUCCAGGGCAACCGUGGCUUUCGAUUGUUACUG 140UGGGAACUGGAGGUAACAGUCUACAGCCAUGGUCGCCCCGCAGC ACGCCCACGCGC miR-132-2GGGCAACCGUGGCUUUCGAUUGUUACUGUGGGAACUGGAGGUAA 141 CAGUCUACAGCCAUGGUCGCCCmiR-133a-1  ACAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUCU 142UCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUAUGCAUUGA miR-133a-2 GGGAGCCAAAUGCUUUGCUAGAGCUGGUAAAAUGGAACCAAAUC 143GACUGUCCAAUGGAUUUGGUCCCCUUCAACCAGCUGUAGCUGUG CAUUGAUGGCGCCG miR-133GCUAGAGCUGGUAAAAUGGAACCAAAUCGCCUCUUCAAUGGAUU 144UGGUCCCCUUCAACCAGCUGUAGC miR-133bccucagaagaaagaugcccccugcucuggcuggucaaacggaaccaaguccgucuuccugagagguu 145ugguccccuucaaccagcuacagcagggcuggcaaugcccaguccuuggaga miR-133b-GCCCCCUGCUCUGGCUGGUCAAACGGAACCAAGUCCGUCUUCCUG 146 smallAGAGGUUUGGUCCCCUUCAACCAGCUACAGCAGGG miR-134-1CAGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUGUGUUCA 147CCCUGUGGGCCACCUAGUCACCAACCCUC miR-134-2AGGGUGUGUGACUGGUUGACCAGAGGGGCAUGCACUGUGUUCAC 148CCUGUGGGCCACCUAGUCACCAACCCU miR-135a-1 AGGCCUCGCUGUUCUCUAUGGCUUUUUAUUCCUAUGUGAUUCUA 149CUGCUCACUCAUAUAGGGAUUGGAGCCGUGGCGCACGGCGGGGA CA miR-135a-2AGAUAAAUUCACUCUAGUGCUUUAUGGCUUUUUAUUCCUAUGUG 150 (miR-135-2)AUAGUAAUAAAGUCUCAUGUAGGGAUGGAAGCCAUGAAAUACAU UGUGAAAAAUCA miR-135CUAUGGCUUUUUAUUCCUAUGUGAUUCUACUGCUCACUCAUAUA 151 GGGAUUGGAGCCGUGGmiR-135bcacucugcuguggccuauggcuuuucauuccuaugugauugcugucccaaacucauguagggcuaa 152aagccaugggcuacagugaggggcgagcucc miR-136-1UGAGCCCUCGGAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAU 153GCUCCAUCAUCGUCUCAAAUGAGUCUUCAGAGGGUUCU miR-136-2GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAUGCUCCAUCAU 154 CGUCUCAAAUGAGUCUUCmiR-137 CUUCGGUGACGGGUAUUCUUGGGUGGAUAAUACGGAUUACGUUG 155UUAUUGCUUAAGAAUACGCGUAGUCGAGG miR-138-1CCCUGGCAUGGUGUGGUGGGGCAGCUGGUGUUGUGAAUCAGGCC 156GUUGCCAAUCAGAGAACGGCUACUUCACAACACCAGGGCCACACC ACACUACAGG miR-138-2CGUUGCUGCAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGCGCA 157UCCUCUUACCCGGCUAUUUCACGACACCAGGGUUGCAUCA miR-138CAGCUGGUGUUGUGAAUCAGGCCGACGAGCAGCGCAUCCUCUUA 158CCCGGCUAUUUCACGACACCAGGGUUG miR-139GUGUAUUCUACAGUGCACGUGUCUCCAGUGUGGCUCGGAGGCUG 159GAGACGCGGCCCUGUUGGAGUAAC miR-140UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAUGGUAG 160GUUACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGA UACCGGGGCACC miR-140asUCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAUGCUGUU 161CUACCACAGGGUAGAACCACGGACAGGA miR-140sCCUGCCAGUGGUUUUACCCUAUGGUAGGUUACGUCAUGCUGUUC 162UACCACAGGGUAGAACCACGGACAGG miR-141-1CGGCCGGCCCUGGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCU 163AAUUGUGAAGCUCCUAACACUGUCUGGUAAAGAUGGCUCCCGGG UGGGUUC miR-141-2GGGUCCAUCUUCCAGUACAGUGUUGGAUGGUCUAAUUGUGAAGC 164UCCUAACACUGUCUGGUAAAGAUGGCCC miR-142ACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAG 165 UGUUUCCUACUUUAUGGAUGmiR-143-1 GCGCAGCGCCCUGUCUCCCAGCCUGAGGUGCAGUGCUGCAUCUCU 166GGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUCAGGAAGAG AGAAGUUGUUCUGCAGC miR-143-2CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGA 167 UGAAGCACUGUAGCUCAGGmiR-144-1 UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAU 168GAGACACUACAGUAUAGAUGAUGUACUAGUCCGGGCACCCCC miR-144-2GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAUGAGACACUA 169 CAGUAUAGAUGAUGUACUAGUCmiR-145-1 CACCUUGUCCUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGC 170UAAGAUGGGGAUUCCUGGAAAUACUGUUCUUGAGGUCAUGGUU miR-145-2CUCACGGUCCAGUUUUCCCAGGAAUCCCUUAGAUGCUAAGAUGG 171GGAUUCCUGGAAAUACUGUUCUUGAG miR-146-1CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGU 172GUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUC UCUGUCAUCGU miR-146-2AGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUCAGACCU 173 GUGAAAUUCAGUUCUUCAGCUmiR-147 AAUCUAAAGACAACAUUUCUGCACACACACCAGACUAUGGAAGC 174CAGUGUGUGGAAAUGCUUCUGCUAGAUU miR-148aGAGGCAAAGUUCUGAGACACUCCGACUCUGAGUAUGAUAGAAGU 175 (miR-148)CAGUGCACUACAGAACUUUGUCUC miR-148bcaagcacgauuagcauuugaggugaaguucuguuauacacucaggcuguggcucucugaaagucag 176ugcaucacagaacuuugucucgaaagcuuucua miR-148b-AAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUACACUCAGG 177 smallCUGUGGCUCUCUGAAAGUCAGUGCAU miR-149-1GCCGGCGCCCGAGCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGU 178CCGAGGAGGGAGGGAGGGACGGGGGCUGUGCUGGGGCAGCUGGA miR-149-2GCUCUGGCUCCGUGUCUUCACUCCCGUGCUUGUCCGAGGAGGGAG 179 GGAGGGAC miR-150-1CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUC 180AGACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC miR-150-2CCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGACCCUGGU 181 ACAGGCCUGGGGGACAGGGmiR-151 UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCU 182ACUAGACUGAAGCUCCUUGAGGACAGG miR-151-2CCUGUCCUCAAGGAGCUUCAGUCUAGUAGGGGAUGAGACAUACU 183AGACUGUGAGCUCCUCGAGGGCAGG miR-152-1UGUCCCCCCCGGCCCAGGUUCUGUGAUACACUCCGACUCGGGCUC 184UGGAGCAGUCAGUGCAUGACAGAACUUGGGCCCGGAAGGACC miR-152-2GGCCCAGGUUCUGUGAUACACUCCGACUCGGGCUCUGGAGCAGUC 185AGUGCAUGACAGAACUUGGGCCCCGG miR-153-1-1CUCACAGCUGCCAGUGUCAUUUUUGUGAUCUGCAGCUAGUAUUC 186UCACUCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGCAGGUGUG GC miR-153-1-2UcUcUcUcUcccUcACAGCUGCCAGUGUCAUUGUCACAAAAGUGAUC 187AUUGGCAGGUGUGGCUGCUGCAUG miR-153-2-1AGCGGUGGCCAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAU 188GAGCCCAGUUGCAUAGUCACAAAAGUGAUCAUUGGAAACUGUG miR-153-2-2CAGUGUCAUUUUUGUGAUGUUGCAGCUAGUAAUAUGAGCCCAGU 189UGCAUAGUCACAAAAGUGAUCAUUG miR-154-1GUGGUACUUGAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUU 190GUGACGAAUCAUACACGGUUGACCUAUUUUUCAGUACCAA miR-154-2GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUUGUGACGAAU 191 CAUACACGGUUGACCUAUUUUUmiR-155 CUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAACUGACU 192CCUACAUAUUAGCAUUAACAG miR-156 =CCUAACACUGUCUGGUAAAGAUGGCUCCCGGGUGGGUUCUCUCG 193 miR-GCAGUAACCUUCAGGGAGCCCUGAAGACCAUGGAGGAC 157 = overlap miR-141 miR-158-GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCC 194 small = miR-AGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGG 192 UAUGUUCGCCUCAAUGCCAGCmiR-159-1- UCCCGCCCCCUGUAACAGCAACUCCAUGUGGAAGUGCCCACUGGU 195 smallUCCAGUGGGGCUGCUGUUAUCUGGGGCGAGGGCCA miR-161-AAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGUGACUGGUCUG 196 smallGGCUACGCUAUGCUGCGGCGCUCGGG miR-163-1b-CAUUGGCCUCCUAAGCCAGGGAUUGUGGGUUCGAGUCCCACCCGG 197 smallGGUAAAGAAAGGCCGAAUU miR-163-3-CCUAAGCCAGGGAUUGUGGGUUCGAGUCCCACCUGGGGUAGAGG 198 smallUGAAAGUUCCUUUUACGGAAUUUUUU miR-162CAAUGUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGG 199 UUGACCAUACUCUACAGUUGmiR-175- GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGAUUGUGC 200 small=miR-AUUGUUUCAAAAUGGUGCCCUAGUGACUACAAAGCCC 224 miR-177-ACGCAAGUGUCCUAAGGUGAGCUCAGGGAGCACAGAAACCUCCA 201 smallGUGGAACAGAAGGGCAAAAGCUCAUU miR-180-CAUGUGUCACUUUCAGGUGGAGUUUCAAGAGUCCCUUCCUGGUU 202 smallCACCGUCUCCUUUGCUCUUCCACAAC miR-181aAGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUCCAAGGAACAUU 203CAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAACCACUGACCGU UGACUGUACCUUGGGGUCCUUAmiR-181b-1ccugugcagagauuauuuuuuaaaaggucacaaucaacauucauugcugucgguggguugaacugu 204guggacaagcucacugaacaaugaaugcaacuguggccccgcuu miR-181b-2cugauggcugcacucaacauucauugcugucgguggguuugagucugaaucaacucacugaucaau 205gaaugcaaacugcggaccaaaca miR-181cCGGAAAAUUUGCCAAGGGUUUGGGGGAACAUUCAACCUGUCGGU 206GAGUUUGGGCAGCUCAGGCAAACCAUCGACCGUUGAGUGGACCC UGAGGCCUGGAAUUGCCAUCCUmiR-182-as GAGCUGCUUGCCUCCCCCCGUUUUUGGCAAUGGUAGAACUCACAC 207UGGUGAGGUAACAGGAUCCGGUGGUUCUAGACUUGCCAACUAUG GGGCGAGGACUCAGCCGGCACmiR-182 UUUUUGGCAAUGGUAGAACUCACACUGGUGAGGUAACAGGAUCC 208GGUGGUUCUAGACUUGCCAACUAUGG miR-183CCGCAGAGUGUGACUCCUGUUCUGUGUAUGGCACUGGUAGAAUU 209CACUGUGAACAGUCUCAGUCAGUGAAUUACCGAAGGGCCAUAAA CAGAGCAGAGACAGAUCCACGAmiR-184-1 CCAGUCACGUCCCCUUAUCACUUUUCCAGCCCAGCUUUGUGACUG 210UAAGUGUUGGACGGAGAACUGAUAAGGGUAGGUGAUUGA miR-184-2CCUUAUCACUUUUCCAGCCCAGCUUUGUGACUGUAAGUGUUGGA 211 CGGAGAACUGAUAAGGGUAGGmiR-185-1 AGGGGGCGAGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCC 212UCCCCAGGGGCUGGCUUUCCUCUGGUCCUUCCCUCCCA miR-185-2AGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCCUCCCCAGG 213 GGCUGGCUUUCCUCUGGUCCUUmiR-186-1 UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUU 214UUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGUUUGAGCU miR-186-2ACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUUUUAUUUUA 215AGCCCAAAGGUGAAUUUUUUGGGAAGU miR-187GGUCGGGCUCACCAUGACACAGUGUGAGACUCGGGCUACAACAC 216AGGACCCGGGGCGCUGCUCUGACCCCUCGUGUCUUGUGUUGCAGC CGGAGGGACGCAGGUCCGCAmiR-188-1 UGCUCCCUCUCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUUUCU 217GAAAACCCCUCCCACAUGCAGGGUUUGCAGGAUGGCGAGCC miR-188-2UCUCACAUCCCUUGCAUGGUGGAGGGUGAGCUUUCUGAAAACCC 218CUCCCACAUGCAGGGUUUGCAGGA miR-189-1CUGUCGAUUGGACCCGCCCUCCGGUGCCUACUGAGCUGAUAUCAG 219UUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAGUCG AGCCCUUGAGCAA miR-189-2CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACU 220GGCUCAGUUCAGCAGGAACAGGAG miR-190-1UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUU 221AAUCCAACUAUAUAUCAAACAUAUUCCUACAGUGUCUUGCC miR-190-2CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAAUCCAACU 222 AUAUAUCAAACAUAUUCCUACAGmiR-191-1 CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCU 223CCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUG CCU miR-191-2AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAU 224UCCAGCUGCGCUUGGAUUUCGUCCCCUGCU miR-192-2/3CCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCA 225GUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGU AUGUUCGCCUCAAUGCCAGmiR-192 GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCC 226AGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGG UAUGUUCGCCUCAAUGCCAGCmiR-193-1 CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGG 227GUGUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG miR-193-2GCUGGGUCUUUGCGGGCGAGAUGAGGGUGUCGGAUCAACUGGCC 228 UACAAAGUCCCAGUmiR-194-1 AUGGUGUUAUCAAGUGUAACAGCAACUCCAUGUGGACUGUGUAC 229CAAUUUCCAGUGGAGAUGCUGUUACUUUUGAUGGUUACCAA miR-194-2GUGUAACAGCAACUCCAUGUGGACUGUGUACCAAUUUCCAGUGG 230 AGAUGCUGUUACUUUUGAUmiR-195-1 AGCUUCCCUGGCUCUAGCAGCACAGAAAUAUUGGCACAGGGAAG 231CGAGUCUGCCAAUAUUGGCUGUGCUGCUCCAGGCAGGGUGGUG miR-195-2UAGCAGCACAGAAAUAUUGGCACAGGGAAGCGAGUCUGCCAAUA 232 UUGGCUGUGCUGCUmiR-196-1 CUAGAGCUUGAAUUGGAACUGCUGAGUGAAUUAGGUAGUUUCAU 233GUUGUUGGGCCUGGGUUUCUGAACACAACAACAUUAAACCACCC GAUUCACGGCAGUUACUGCUCCmiR-196a-1 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACA 234CAACAACAUUAAACCACCCGAUUCAC miR-196a-2UGCUCGCUCAGCUGAUCUGUGGCUUAGGUAGUUUCAUGUUGUUG 235 (miR-196-2)GGAUUGAGUUUUGAACUCGGCAACAAGAAACUGCCUGAGUUACA UCAGUCGGUUUUCGUCGAGGGCmiR-196 GUGAAUUAGGUAGUUUCAUGUUGUUGGGCCUGGGUUUCUGAACA 236CAACAACAUUAAACCACCCGAUUCAC miR-196bacuggucggugauuuagguaguuuccuguuguugggauccaccuuucucucgacagcacgacacug 237ccuucauuacuucaguug miR-197 GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCA238 CCCUUCACCACCUUCUCCACCCAGCAUGGCC miR-197-2GUGCAUGUGUAUGUAUGUGUGCAUGUGCAUGUGUAUGUGUAUGA 239 GUGCAUGCGUGUGUGCmiR-198 UCAUUGGUCCAGAGGGGAGAUAGGUUCCUGUGAUUUUUCCUUCU 240UCUCUAUAGAAUAAAUGA miR-199a-1GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGU 241GUACAGUAGUCUGCACAUUGGUUAGGC miR-199a-2AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACU 242ACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGU UAGACUGGGCAAGGGAGAGCAmiR-199b CCAGAGGACACCUCCACUCCGUCUACCCAGUGUUUAGACUAUCUG 243UUCAGGACUCCCAAAUUGUACAGUAGUCUGCACAUUGGUUAGGC UGGGCUGGGUUAGACCCUCGGmiR-199s GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGU 244GUACAGUAGUCUGCACAUUGGUUAGGC miR-200aGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUGGAGUCAGGUCUC 245UAAUACUGCCUGGUAAUGAUGACGGC miR-200bCCAGCUCGGGCAGCCGUGGCCAUCUUACUGGGCAGCAUUGGAUG 246GAGUCAGGUCUCUAAUACUGCCUGGUAAUGAUGACGGCGGAGCC CUGCACG miR-200ccccucgucuuacccagcaguguuugggugcgguugggagucucuaauacugccggguaaugaugga 247gg miR-202 GUUCCUUUUUCCUAUGCAUAUACUUCUUUGAGGAUCUGGCCUAA 248AGAGGUAUAGGGCAUGGGAAGAUGGAGC miR-203GUGUUGGGGACUCGCGCGCUGGGUCCAGUGGUUCUUAACAGUUC 249AACAGUUCUGUAGCGCAAUUGUGAAAUGUUUAGGACCACUAGAC CCGGCGGGCGCGGCGACAGCGAmiR-204 GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCA 250UCCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGG ACGUUCAAUUGUCAUCACUGGCmiR-205 AAAGAUCCUCAGACAAUCCAUGUGCUUCUCUUGUCCUUCAUUCCA 251CCGGAGUCUGUCUCAUACCCAACCAGAUUUCAGUGGAGUGAAGU UCAGGAGGCAUGGAGCUGACAmiR-206-1 UGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUAC 252UUUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG miR-206-2AGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACUUUGCUAU 253GGAAUGUAAGGAAGUGUGUGGUUUU miR-208UGACGGGCGAGCUUUUGGCCCGGGUUAUACCUGAUGCUCACGUA 254UAAGACGAGCAAAAAGCUUGUUGGUCA miR-210ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCAC 255ACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCU GUGCCUGGGCAGCGCGACCCmiR-211 UCACCUGGCCAUGUGACUUGUGGGCUUCCCUUUGUCAUCCUUCGC 256CUAGGGCUCUGAGCAGGGCAGGGACAGCAAAGGGGUGCUCAGUU GUCACUUCCCACAGCACGGAGmiR-212 CGGGGCACCCCGCCCGGACAGCGCGCCGGCACCUUGGCUCUAGAC 257UGCUUACUGCCCGGGCCGCCCUCAGUAACAGUCUCCAGUCACGGC CACCGACGCCUGGCCCCGCCmiR-213-2 CCUGUGCAGAGAUUAUUUUUUAAAAGGUCACAAUCAACAUUCAU 258UGCUGUCGGUGGGUUGAACUGUGUGGACAAGCUCACUGAACAAU GAAUGCAACUGUGGCCCCGCUUmiR-213 GAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUCGGUGAG 259UUUGGAAUUAAAAUCAAAACCAUCGACCGUUGAUUGUACCCUAU GGCUAACCAUCAUCUACUCCmiR-214 GGCCUGGCUGGACAGAGUUGUCAUGUGUCUGCCUGUCUACACUU 260GCUGUGCAGAACAUCCGCUCACCUGUACAGCAGGCACAGACAGGC AGUCACAUGACAACCCAGCCUmiR-215 AUCAUUCAGAAAUGGUAUACAGGAAAAUGACCUAUGAAUUGACA 261GACAAUAUAGCUGAGUUUGUCUGUCAUUUCUUUAGGCCAAUAUU CUGUAUGACUGUGCUACUUCAAmiR-216 GAUGGCUGUGAGUUGGCUUAAUCUCAGCUGGCAACUGUGAGAUGUUCAUACAAUCCCUCACAGUGGUCUCUGGGAUUAUGCUAAACAG 262 AGCAAUUUCCUAGCCCUCACGAmiR-217 AGUAUAAUUAUUACAUAGUUUUUGAUGUCGCAGAUACUGCAUCA 263GGAACUGAUUGGAUAAGAAUCAGUCACCAUCAGUUCCUAAUGCA UUGCCUUCAGCAUCUAAACAAGmiR-218-1 GUGAUAAUGUAGCGAGAUUUUCUGUUGUGCUUGAUCUAACCAUG 264UGGUUGCGAGGUAUGAGUAAAACAUGGUUCCGUCAAGCACCAUG GAACGUCACGCAGCUUUCUACAmiR-218-2 GACCAGUCGCUGCGGGGCUUUCCUUUGUGCUUGAUCUAACCAUG 265UGGUGGAACGAUGGAAACGGAACAUGGUUCUGUCAAGCACCGCG GAAAGCACCGUGCUCUCCUGCAmiR-219 CCGCCCCGGGCCGCGGCUCCUGAUUGUCCAAACGCAAUUCUCGAG 266UCUAUGGCUCCGGCCGAGAGUUGAGUCUGGACGUCCCGAGCCGCC GCCCCCAAACCUCGAGCGGGmiR-219-1ccgccccgggccgcggcuccugauuguccaaacgcaauucucgagucuauggcuccggccgagaguu 267gagucuggacgucccgagccgccgcccccaaaccucgagcggg miR-219-2acucaggggcuucgccacugauuguccaaacgcaauucuuguacgagucugcggccaaccgagaauu 268guggcuggacaucuguggcugagcuccggg miR-220GACAGUGUGGCAUUGUAGGGCUCCACACCGUAUCUGACACUUUG 269GGCGAGGGCACCAUGCUGAAGGUGUUCAUGAUGCGGUCUGGGAA CUCCUCACGGAUCUUACUGAUGmiR-221 UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAU 270UUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCA GGCUACCUGGAAACAUGUUCUCmiR-222 GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAGCCAGUG 271UAGAUCCUGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGG UCUCUGAUGGCAUCUUCUAGCUmiR-223 CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAAGCUGAG 272UUGGACACUCCAUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCA AGUGCGGCACAUGCUUACCAGmiR-224 GGGCUUUCAAGUCACUAGUGGUUCCGUUUAGUAGAUGAUUGUGC 273AUUGUUUCAAAAUGGUGCCCUAGUGACUACAAAGCCC miR-294-1CAAUCUUCCUUUAUCAUGGUAUUGAUUUUUCAGUGCUUCCCUUU 274 (chr16)UGUGUGAGAGAAGAUA miR-296aggacccuuccagagggcccccccucaauccuguugugccuaauucagaggguuggguggaggcuc  275uccugaagggcucu miR-299aagaaaugguuuaccgucccacauacauuuugaauauguaugugggaugguaaaccgcuucuu 276miR-301acugcuaacgaaugcucugacuuuauugcacuacuguacuuuacagcuagcagugcaauaguauug  277ucaaagcaucugaaagcagg miR-302accaccacuuaaacguggauguacuugcuuugaaacuaaagaaguaagugcuuccauguuuugguga  278ugg miR-302bgcucccuucaacuuuaacauggaagugcuuucugugacuuuaaaaguaagugcuuccauguuuua 279guaggagu miR-302cccuuugcuuuaacauggggguaccugcugugugaaacaaaaguaagugcuuccauguuucagugg 280agg miR-302dccucuacuuuaacauggaggcacuugcugugacaugacaaaaauaagugcuuccauguuugagugu 281gg miR-320gcuucgcuccccuccgccuucucuucccgguucuucccggagucgggaaaagcuggguugagagg 282gcgaaaaaggaugaggu miR-321uuggccuccuaagccagggauuguggguucgaguccacccgggguaaagaaaggccga 283 miR-323uugguacuuggagagaggugguccguggcgcguucgcuuuauuuauggcgcacauuacacggucg 284accucuuugcaguaucuaauc miR-324cugacuaugccuccccgcauccccuagggcauugguguaaagcuggagacccacugccccaggugc 285miR-325auacagugcuugguuccuaguagguguccaguaaguguuugugacauaauuuguuuauugaggac 286cuccuaucaaucaagcacugugcuaggcucugg miR-326cucaucugucuguugggcuggaggcagggccuuugugaaggcggguggugcucagaucgccucug 287ggcccuuccuccagccccgaggcggauuca miR-328uggagugggggggcaggaggggcucagggagaaagugcauacagccccuggcccucucugcccuu 288ccgucccug miR-330cuuuggcgaucacugccucucugggccugugucuuaggcucugcaagaucaaccgagcaaagcaca 289cggccugcagagaggcagcgcucugccc miR-331gaguuugguuuuguuuggguuuguucuagguauggucccagggaucccagaucaaaccaggcccc 290ugggccuauccuagaaccaaccuaagcuc muR-335uguuuugagcgggggucaagagcaauaacgaaaaauguuugucauaaaccguuuuucauuauugc 291uccugaccuccucucauuugcuauauuca miR-337guagucaguaguuggggggugggaacggcuucauacaggaguugaugcacaguuauccagcuccu 292auaugaugccuuucuucauccccuucaa miR-338ucuccaacaauauccuggugcugagugaugacucaggcgacuccagcaucagugauuuuguugaag 293 amiR-339cggggcggccgcucucccuguccuccaggagcucacgugugccugccugugagcgccucgacgaca 294gagccggcgccugccccagugucugcgc miR-340uuguaccuggugugauuauaaagcaaugagacugauugucauaugucguuugugggauccgucuc 295aguuacuuuauagccauaccugguaucuua miR-342gaaacugggcucaaggugaggggugcuaucugugauugagggacaugguuaauggaauugucuca 296cacagaaaucgcacccgucaccuuggccuacuua miR-345acccaaacccuaggucugcugacuccuaguccagggcucgugauggcuggugggcccugaacgagg 297ggucuggaggccuggguuugaauaucgacagc miR-346gucugucugcccgcaugccugccucucuguugcucugaaggaggcaggggcugggccugcagcug 298ccugggcagagcggcuccugc miR-367ccauuacuguugcuaauaugcaacucuguugaauauaaauuggaauugcacuuuagcaauggugau 299gg miR-368aaaagguggauauuccuucuauguuuauguuauuuagguuaaacauagaggaaauuccacguuu 300 umiR-369uugaagggagaucgaccguguuauauucgcuuuauugacuucgaauaauacaugguugaucuuuu 301cucag miR-370agacagagaagccaggucacgucucugcaguuacacagcucacgagugccugcugggguggaaccu 302ggucugucu miR-371guggcacucaaacugugggggcacuuucugcucucuggugaaagugccgccaucuuuugaguguu 303 acmiR-372gugggccucaaauguggagcacuauucugauguccaaguggaaagugcugcgacauuugagcguc 304 acmiR-373 gggauacucaaaaugggggcgcuuuccuuuuugucuguacugggaagugcuucgauuuuggggu305 guccc miR-374uacaucggccauuauaauacaaccugauaaguguuauagcacuuaucagauuguauuguaauuguc 306ugugua miR-hes1 AUGGAGCUGCUCACCCUGUGGGCCUCAAAUGUGGAGGAACUAUU 307CUGAUGUCCAAGUGGAAAGUGCUGCGACAUUUGAGCGUCACCGG UGACGCCCAUAUCA miR-hes2GCAUCCCCUCAGCCUGUGGCACUCAAACUGUGGGGGCACUUUCU 308GCUCUCUGGUGAAAGUGCCGCCAUCUUUUGAGUGUUACCGCUUG AGAAGACUCAACC miR-hes3CGAGGAGCUCAUACUGGGAUACUCAAAAUGGGGGCGCUUUCCUUUUUGUCUGUUACUGGGAAGUGCUUCGAUUUUGGGGUGUCCCUG UUUGAGUAGGGCAUC

An underlined sequence within a precursor sequence corresponds to amature processed miR transcript (see Table 1b). Some precursor sequenceshave two underlined sequences denoting two different mature miRs thatare derived from the same precursor. All sequences are human.

TABLE 1b Human Mature microRNA Sequences. Mature miRNAMature miRNA Sequence SEQ ID Corresponding precursor microRNA(s); Name(5′ to 3′) NO. see Table 1a let-7a ugagguaguagguuguauaguu 310let-7a-1; let-7a-2; let-7a-3; let-7a-4 let-7b ugagguaguagguugugugguu 311let-7b let-7c ugagguaguagguuguaugguu 312 let-7c let-7dagagguaguagguugcauagu 313 let-7dlet-7d-v1 let-7e ugagguaggagguuguauagu314 let-7e let-7f ugagguaguagauuguauaguu 315let-7f; let-7f-2-1; let-7f-2-2 let-7g ugagguaguaguuuguacagu 316 let-7glet-7i ugagguaguaguuugugcu 317 let-7i miR-1 uggaauguaaagaaguaugua 318miR-1b; miR-1b-1; miR-1b-2 miR-7 uggaagacuagugauuuuguu 319miR-7-1; miR-7-1a; miR-7-2; miR-7-3 miR-9 ucuuugguuaucuagcuguauga 320miR-9-1; miR-9-2; miR-9-3 miR-9* uaaagcuagauaaccgaaagu 321miR-9-1; miR-9-2; miR-9-3 miR-10a uacccuguagauccgaauuugug 322 miR-10amiR-10b uacccuguagaaccgaauuugu 323 miR-10b miR-15auagcagcacauaaugguuugug 324 miR-15a; miR-15a-2 miR-15buagcagcacaucaugguuuaca 325 miR-15b miR-16 uagcagcacguaaauauuggcg 326miR-16-1; miR-16-2; miR-16-13 miR-17-5p caaagugcuuacagugcagguagu 327miR-17 miR-17-3p acugcagugaaggcacuugu 328 miR-17 miR-18uaaggugcaucuagugcagaua 329 miR-18; miR-18-13 miR-19augugcaaaucuaugcaaaacuga 330 miR-19a; miR-19a-13 miR-19bugugcaaauccaugcaaaacuga 331 miR-19b-1; miR-19b-2 miR-20uaaagugcuuauagugcaggua 332 miR-20 (miR-20a) miR-21uagcuuaucagacugauguuga 333 miR-21; miR-21-17 miR-22aagcugccaguugaagaacugu 334 miR-22 miR-23a aucacauugccagggauuucc 335miR-23a miR-23b aucacauugccagggauuaccac 336 miR-23b miR-24uggcucaguucagcaggaacag 337 miR-24-1; miR-24-2; miR-24-19; miR-24-9miR-25 cauugcacuugucucggucuga 338 miR-25 miR-26a uucaaguaauccaggauaggcu339 miR-26a; miR-26a-1; miR-26a-2 miR-26b uucaaguaauucaggauaggu 340miR-26b miR-27a uucacaguggcuaaguuccgcc 341 miR-27a miR-27buucacaguggcuaaguucug 342 miR-27b-1; miR-27b-2 miR-28aaggagcucacagucuauugag 343 miR-28 miR-29a cuagcaccaucugaaaucgguu 344miR-29a-2; miR-29a miR-29b uagcaccauuugaaaucagu 345 miR-29b-1; miR-29b-2miR-29c uagcaccauuugaaaucgguua 346 miR-29c miR-30a-5puguaaacauccucgacuggaagc 347 miR-30a miR-30a-3p cuuucagucggauguuugcagc348 miR-30a miR-30b uguaaacauccuacacucagc 349 miR-30b-1; miR-30b-2miR-30c uguaaacauccuacacucucagc 350 miR-30c miR-30duguaaacauccccgacuggaag 351 miR-30d miR-30e uguaaacauccuugacugga 352miR-30e miR-31 ggcaagaugcuggcauagcug 353 miR-31 miR-32uauugcacauuacuaaguugc 354 miR-32 miR-33 gugcauuguaguugcauug 355miR-33; miR-33b miR-34a uggcagugucuuagcugguugu 356 miR-34a miR-34baggcagugucauuagcugauug 357 miR-34b miR-34c aggcaguguaguuagcugauug 358miR-34c miR-92 uauugcacuugucccggccugu 359 miR-92-2; miR-92-1 miR-93aaagugcuguucgugcagguag 360 miR-93-1; miR-93-2 miR-95uucaacggguauuuauugagca 361 miR-95 miR-96 uuuggcacuagcacauuuuugc 362miR-96 miR-98 ugagguaguaaguuguauuguu 363 miR-98 miR-99aaacccguagauccgaucuugug 364 miR-99a miR-99b cacccguagaaccgaccuugcg 365miR-99b miR-100 uacaguacugugauaacugaag 366 miR-100 miR-101uacaguacugugauaacugaag 367 miR-101-1; miR-101-2 miR-103acagcauuguacagggcuauga 368 miR-103-1 miR-105 ucaaaugcucagacuccugu 369miR-105 miR-106-a aaaagugcuuacagugcagguagc 370 miR-106-a miR-106-buaaagugcugacagugcagau 371 miR-106-b miR-107 agcagcauuguacagggcuauca 372miR-107 miR-122a uggagugugacaaugguguuugu 373 miR-122a-1; miR-122a-2miR-124a uuaaggcacgcggugaaugcca 374 miR-124a-1; miR-124a-2; miR-124a-3miR-125a ucccugagacccuuuaaccugug 375 miR-125a-1; miR-125a-2 miR-125bucccugagacccuaacuuguga 376 miR-125b-1; miR-125b-2 miR-126*cauuauuacuuuugguacgc 377 miR-126-1; miR-126-2 miR-126ucguaccgugaguaauaaugc 378 miR-126-1; miR-126-2 miR-127ucggauccgucugagcuuggcu 379 miR-127-1; miR-127-2 miR-128aucacagugaaccggucucuuuu 380 miR-128; miR-128a miR-128bucacagugaaccggucucuuuc 381 miR-128b miR-129 cuuuuugcggucugggcuugc 382miR-129-1; miR-129-2 miR-130a cagugcaauguuaaaagggc 383 miR-130a miR-130bcagugcaaugaugaaagggcau 384 miR-130b miR-132 uaacagucuacagccauggucg 385miR-132-1 miR-133a uugguccccuucaaccagcugu 386 miR-133a-1; miR-133a-2miR-133b uugguccccuucaaccagcua 387 miR-133b miR-134ugugacugguugaccagaggg 388 miR-134-1; miR-134-2 miR-135auauggcuuuuuauuccuauguga 389 miR-135a; miR-135a-2 (miR-135-2) miR-135buauggcuuuucauuccuaugug 390 miR-135b miR-136 acuccauuuguuuugaugaugga 391miR-136-1; miR-136-2 miR-137 uauugcuuaagaauacgcguag 392 miR-137 miR-138agcugguguugugaauc 393 miR-138-1; miR-138-2 miR-139 ucuacagugcacgugucu394 miR-139 miR-140 agugguuuuacccuaugguag 395miR-140-1; miR-140as; miR-140s miR-141 aacacugucugguaaagaugg 396miR-141-1; miR-141-2 miR-142-3p uguaguguuuccuacuuuaugga 397 miR-142miR-142-5p cauaaaguagaaagcacuac 398 miR-142 miR-143ugagaugaagcacuguagcuca 399 miR-143-1 miR-144 uacaguauagaugauguacuag 400miR-144-1; miR-144-2 miR-145 guccaguuuucccaggaaucccuu 401miR-145-1; miR-145-2 miR-146 ugagaacugaauuccauggguu 402miR-146-1; miR-146-2 miR-147 guguguggaaaaugcuucugc 403 miR-147 miR-148aucagugcacuacagaacuuugu 404 miR-148a (miR-148) miR-148bucagugcaucacagaacuuugu 405 miR-148b miR-149 ucuggcuccgugucuucacucc 406miR-149 miR-150 ucucccaacccuuguaccagug 407 miR-150-1; miR-150-2 miR-151acuagacugaagcuccuugagg 408 miR-151 miR-152 ucagugcaugacagaacuugg 409miR-152-1; miR-152-2 miR-153 uugcauagucacaaaaguga 410miR-153-1-1; miR-153-1-2; miR-153-2-1; miR-153-2-2 miR-154uagguuauccguguugccuug 411 miR-154-1; miR-154-2 miR-154*aaucauacacgguugaccuauu 412 miR-154-1; miR-154-2 miR-155uuaaugcuaaucgugauagggg 413 miR-155 miR-181a aacauucaacgcugucggugagu 414miR-181a miR-181b aacauucauugcugucgguggguu 415 miR-181b-1; miR-181b-2miR-181c aacauucaaccugucggugagu 416 miR-181c miR-182uuuggcaaugguagaacucaca 417 miR-182; miR-182as miR-182*ugguucuagacuugccaacua 418 miR-182; miR-182as miR-183uauggcacugguagaauucacug 419 miR-183 miR-184 uggacggagaacugauaagggu 420miR-184-1; miR-184-2 miR-185 uggagagaaaggcaguuc 421 miR-185-1; miR-185-2miR-186 caaagaauucuccuuuugggcuu 422 miR-186-1; miR-186-2 miR-187ucgugucuuguguugcagccg 423 miR-187 miR-188 caucccuugcaugguggagggu 424miR-188 miR-189 gugccuacugagcugauaucagu 425 miR-189-1; miR-189-2 miR-190ugauauguuugauauauuaggu 426 miR-190-1; miR-190-2 miR-191caacggaaucccaaaagcagcu 427 miR-191-1; miR-191-2 miR-192cugaccuaugaauugacagcc 428 miR-192 miR-193 aacuggccuacaaagucccag 429miR-193-1; miR-193-2 miR-194 uguaacagcaacuccaugugga 430miR-194-1; miR-194-2 miR-195 uagcagcacagaaauauuggc 431miR-195-1; miR-195-2 miR-196a uagguaguuucauguuguugg 432miR-196a; miR-196a-2 (miR196-2) miR-196b uagguaguuuccuguuguugg 433miR-196b miR-197 uucaccaccuucuccacccagc 434 miR-197 miR-198gguccagaggggagauagg 435 miR-198 miR-199a cccaguguucagacuaccuguuc 436miR-199a-1; miR-199a-2 miR-199a* uacaguagucugcacauugguu 437miR-199a-1; miR-199a-2; miR-199s; miR-199b miR-199bcccaguguuuagacuaucuguuc 438 miR-199b miR-200a uaacacugucugguaacgaugu 439miR-200a miR-200b cucuaauacugccugguaaugaug 440 miR-200b miR-200caauacugccggguaaugaugga 441 miR-200c miR-202 agagguauagggcaugggaaga 442miR-202 miR-203 gugaaauguuuaggaccacuag 443 miR-203 miR-204uucccuuugucauccuaugccu 444 miR-204 miR-205 uccuucauuccaccggagucug 445miR-205 miR-206 uggaauguaaggaagugugugg 446 miR-206-1; miR-206-2 miR-208auaagacgagcaaaaagcuugu 447 miR-208 miR-210 cugugcgugugacagcggcug 448miR-210 miR-211 uucccuuugucauccuucgccu 449 miR-211 miR-212uaacagucuccagucacggcc 450 miR-212 miR-213 accaucgaccguugauuguacc 451miR-213 miR-214 acagcaggcacagacaggcag 452 miR-214 miR-215augaccuaugaauugacagac 453 miR-215 miR-216 uaaucucagcuggcaacugug 454miR-216 miR-217 uacugcaucaggaacugauuggau 455 miR-217 miR-218uugugcuugaucuaaccaugu 456 miR-218-1; miR-218-2 miR-219ugauuguccaaacgcaauucu 457 miR-219; miR-219-1; miR-219-2 miR-220ccacaccguaucugacacuuu 458 miR-220 miR-221 agcuacauugucugcuggguuuc 459miR-221 miR-222 agcuacaucuggcuacugggucuc 460 miR-222 miR-223ugucaguuugucaaauacccc 461 miR-223 miR-224 caagucacuagugguuccguuua 462miR-224 miR-296 agggcccccccucaauccugu 463 miR-296 miR-299ugguuuaccgucccacauacau 464 miR-299 miR-301 cagugcaauaguauugucaaagc 465miR-301 miR-302a uaagugcuuccauguuuugguga 466 miR-302a miR-302b*acuuuaacauggaagugcuuucu 467 miR-302b miR-302b uaagugcuuccauguuuuaguag468 miR-302b miR-302c* uuuaacauggggguaccugcug 469 miR-302c miR-302cuaagugcuuccauguuucagugg 470 miR-302c miR-302d uaagugcuuccauguuugagugu471 miR-302d miR-320 aaaagcuggguugagagggcgaa 472 miR-320 miR-321uaagccagggauuguggguuc 473 miR-321 miR-323 gcacauuacacggucgaccucu 474miR-323 miR-324-5p cgcauccccuagggcauuggugu 475 miR-324 miR-324-3pccacugccccaggugcugcugg 476 miR-324 miR-325 ccuaguagguguccaguaagu 477miR-325 miR-326 ccucugggcccuuccuccag 478 miR-326 miR-328cuggcccucucugcccuuccgu 479 miR-328 miR-330 gcaaagcacacggccugcagaga 480miR-330 miR-331 gccccugggccuauccuagaa 481 miR-331 miR-335ucaagagcaauaacgaaaaaugu 482 miR-335 miR-337 uccagcuccuauaugaugccuuu 483miR-337 miR-338 uccagcaucagugauuuuguuga 484 miR-338 miR-339ucccuguccuccaggagcuca 485 miR-339 miR-340 uccgucucaguuacuuuauagcc 486miR-340 miR-342 ucucacacagaaaucgcacccguc 487 miR-342 miR-345ugcugacuccuaguccagggc 488 miR-345 miR-346 ugucugcccgcaugccugccucu 489miR-346 miR-367 aauugcacuuuagcaaugguga 490 miR-367 miR-368acauagaggaaauuccacguuu 491 miR-368 miR-369 aauaauacaugguugaucuuu 492miR-369 miR-370 gccugcugggguggaaccugg 493 miR-370 miR-371gugccgccaucuuuugagugu 494 miR-371 miR-372 aaagugcugcgacauuugagcgu 495miR-372 miR-373* acucaaaaugggggcgcuuucc 496 miR-373 miR-373gaagugcuucgauuuuggggugu 497 miR-373 miR-374 uuauaauacaaccugauaagug 498miR-374

The level of at least one miR gene product can be measured in cells of abiological sample obtained from the subject. For example, a tissuesample can be removed from a subject suspected of having pancreaticcancer by conventional biopsy techniques. In another embodiment, a bloodsample can be removed from the subject, and white blood cells can beisolated for DNA extraction by standard techniques. The blood or tissuesample is preferably obtained from the subject prior to initiation ofradiotherapy, chemotherapy or other therapeutic treatment. Acorresponding control tissue or blood sample, or a control referencesample, can be obtained from unaffected tissues of the subject, from anormal human individual or population of normal individuals, or fromcultured cells corresponding to the majority of cells in the subject'ssample. The control tissue or blood sample is then processed along withthe sample from the subject, so that the levels of miR gene productproduced from a given miR gene in cells from the subject's sample can becompared to the corresponding miR gene product levels from cells of thecontrol sample. Alternatively, a reference sample can be obtained andprocessed separately (e.g., at a different time) from the test sampleand the level of a miR gene product produced from a given miR gene incells from the test sample can be compared to the corresponding miR geneproduct level from the reference sample.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “upregulated”). As used herein, expression of a miR gene product is“upregulated” when the amount of miR gene product in a cell or tissuesample from a subject is greater than the amount of the same geneproduct in a control cell or tissue sample. In another embodiment, thelevel of the at least one miR gene product in the test sample is lessthan the level of the corresponding miR gene product in the controlsample (i.e., expression of the miR gene product is “downregulated”). Asused herein, expression of a miR gene is “downregulated” when the amountof miR gene product produced from that gene in a cell or tissue samplefrom a subject is less than the amount produced from the same gene in acontrol cell or tissue sample. The relative miR gene expression in thecontrol and normal samples can be determined with respect to one or moreRNA expression standards. The standards can comprise, for example, azero miR gene expression level, the miR gene expression level in astandard cell line, the miR gene expression level in unaffected tissuesof the subject, or the average level of miR gene expression previouslyobtained for a population of normal human controls.

An alteration (i.e., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of pancreatic cancer in the subject. In one embodiment,the level of the at least one miR gene product in the test sample isgreater than the level of the corresponding miR gene product in thecontrol sample. miR gene products having higher expression levels inpancreatic cancer than normal pancreatic tissue are described herein(see, e.g., Exemplification). In one embodiment, the at least one miRgene product is selected from the group consisting of miR-103-2,miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a,miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2,miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25,miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b,miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a,miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2,miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v,miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3,miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375 and a combination thereof. In anotherembodiment, the at least one miR gene product is selected from the groupconsisting of miR-103, miR-107 and a combination thereof. In yet anotherembodiment, the at least one miR gene product is selected from the groupconsisting of miR-23a, miR-26b, miR-192, miR-342 and a combinationthereof.

In one embodiment, the level of the at least one miR gene product in thetest sample is less than the level of the corresponding miR gene productin the control sample. miR gene products having lower expression levelsin pancreatic cancer than normal pancreatic tissue are described herein(see, e.g., Exemplification). In one embodiment, the at least one miRgene product is selected from the group consisting of miR-326, miR-155,miR-339, miR-34c, miR-345, miR-152, miR-372, miR-128a and a combinationthereof. In another embodiment, the at least one miR gene product ismiR-155.

In one embodiment, the at least one miR gene product is selected fromthe group consisting of miR-103, is miR-107, miR-155 and a combinationthereof. In another embodiment, the at least one miR gene product ismiR-103, which is upregulated in the test sample, as compared to thecontrol sample. In yet another embodiment, the at least one miR geneproduct is miR-107, which is upregulated in the test sample, as comparedto the control sample. In still another embodiment, the at least one miRgene product is miR-155, which is downregulated in the test sample, ascompared to the control sample. In a particular embodiment, all three ofthese miRs (miR-103, miR-107 and miR-155) are compared to thecorresponding miRs in the control sample. As described and exemplifiedherein, the expression of miR-103 and miR-107, associated with lack ofexpression of miR-155, discriminates pancreatic tumors from normalpancreas.

In one embodiment, the pancreatic cancer that is diagnosed is apancreatic endocrine tumor (PET). In another embodiment, the pancreaticcancer that is diagnosed is a pancreatic exocrine tumor (e.g., anadenocarcinoma). In yet another embodiment, the pancreatic cancer thatis diagnosed is selected from the group consisting of a pancreaticendocrine tumor (PET) and a pancreatic exocrine tumor (e.g., anadenocarcinoma). In a particular embodiment, the pancreatic cancer thatis diagnosed is selected from the group consisting of an acinar cellcarcinoma (PACC) and an insulinoma. In yet another embodiment, thepancreatic cancer that is diagnosed is selected from the groupconsisting of a pancreatic endocrine tumor (PET), a pancreatic acinarcell carcinoma (PACC) and an insulinoma. In still another embodiment,the diagnostic method can be used to diagnose any type of pancreaticcancer.

In one embodiment, the invention is a method of diagnosing whether asubject has, or is at risk for developing, pancreatic acinar cellcarcinoma (PACC). In this method, the level of at least one miR geneproduct in a test sample from the subject is compared to the level of acorresponding miR gene product in a control sample. An alteration (e.g.,an increase, a decrease) in the level of the miR gene product in thetest sample, relative to the level of a corresponding miR gene productin a control sample, is indicative of the subject either having, orbeing at risk for developing, PACC. In one embodiment, the level of theat least one miR gene product in the test sample is greater than thelevel of the corresponding miR gene product in the control sample. Inanother embodiment, the at least one miR gene product that isupregulated is selected from the group consisting of miR-103-2, miR-25,miR-200c, miR-335, miR-21, miR-103-1, miR-92-1, miR-181b-2, miR-191,miR-93, miR-26a-1, miR-17, miR-20, miR-107, miR-26b, miR-215, miR-92-2,miR-192, miR-342, miR-100, miR-3p21-v, miR-106a, miR-15a, miR-23a,miR-181b-1, miR-128b, miR-106b, miR-194-1, miR-219-1, miR-242 and acombination thereof. In yet another embodiment, the level of the atleast one miR gene product in the test sample is less than the level ofthe corresponding miR gene product in the control sample. In stillanother embodiment, the at least one miR gene product that isdownregulated is selected from the group consisting of miR-218-2,miR-339, miR-326, miR-34c, miR-152, miR-138-2, miR-128a and acombination thereof.

In one embodiment, the invention is a method of diagnosing the type ofpancreatic cancer that a subject has. In this method, the level of atleast one miR gene product in a test sample from the subject is comparedto the level of a corresponding miR gene product in a control sample. Analteration (e.g., an increase, a decrease) in the level of the miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of the type ofpancreatic cancer.

In a particular embodiment, the type of pancreatic cancer that isdiagnosed is selected from the group consisting of a pancreaticendocrine tumor (PET) and a pancreatic acinar cell carcinoma (PACC). Inanother embodiment, the level of the at least one miR gene product inthe test sample is greater than the level of the corresponding miR geneproduct in the control sample. In another embodiment, the type ofpancreatic cancer is a pancreatic endocrine tumor (PET) and the at leastone miR gene product that is upregulated is selected from the groupconsisting of miR-125a, miR-99a, miR-99b, miR-125b-1, miR-342, miR-130a,miR-100, miR-132, miR-129-2, miR-125b-2 and a combination thereof. Inyet another embodiment, the level of the at least one miR gene productin the test sample is less than the level of the corresponding miR geneproduct in the control sample. In still another embodiment, the type ofpancreatic cancer is a pancreatic acinar cell carcinoma (PACC) and theat least one miR gene product that is downregulated is selected from thegroup consisting of miR-125a, miR-99a, miR-99b, miR-125b-1, miR-342,miR-130a, miR-100, miR-132, miR-129-2, miR-125b-2 and a combinationthereof. As described herein, the expression of particular miR geneproducts can distinguish between PET and PACC (see, e.g.,Exemplification).

In one embodiment, the type of pancreatic cancer that is diagnosed isselected from the group consisting of a well-differentiated endocrinecarcinoma (WDEC) and a pancreatic acinar cell carcinoma (PACC). Inanother embodiment, the level of the at least one miR gene product inthe test sample is greater than the level of the corresponding miR geneproduct in the control sample. In yet another embodiment, the type ofpancreatic cancer is a well-differentiated endocrine carcinoma (WDEC)and the at least one miR gene product that is upregulated is selectedfrom the group consisting of miR-125a, miR-99a, miR-132 and acombination thereof. In another embodiment, the level of the at leastone miR gene product in the test sample is less than the level of thecorresponding miR gene product in the control sample. In still anotherembodiment, the type of pancreatic cancer is a well-differentiatedendocrine carcinoma (WDEC) and the at least one miR gene product that isdownregulated is miR-148a. As described herein, the expression ofparticular miR gene products can distinguish between WDEC and PACC (see,e.g., Exemplification).

In one embodiment, the type of pancreatic cancer that is diagnosed isselected from the group consisting of an insulinoma and anon-functioning pancreatic endocrine tumor (NF-PET). In one embodiment,the level of the at least one miR gene product in the test sample isgreater than the level of the corresponding miR gene product in thecontrol sample. In another embodiment, the type of pancreatic cancer isan insulinoma and the at least one miR gene product that is upregulatedis selected from the group consisting of miR-204, miR-203, miR-211 and acombination thereof. As described herein, the expression of particularmiR gene products can distinguish between WDEC and PACC (see, e.g.,Exemplification).

The invention also provides methods of determining the prognosis of asubject with pancreatic cancer. In this method, the level of at leastone miR gene product, which is associated with a particular prognosis inpancreatic cancer (e.g., a good or positive prognosis, a poor or adverseprognosis), is measured in a test sample from the subject. An alteration(e.g., an increase, a decrease) in the level of the miR gene product inthe test sample, relative to the level of a corresponding miR geneproduct in a control sample, is indicative of the subject having apancreatic cancer with a particular prognosis. In one embodiment, themiR gene product is associated with an adverse (i.e., poor) prognosis.Examples of an adverse prognosis include, but are not limited to, lowsurvival rate and rapid disease progression. In one embodiment, thelevel of the at least one miR gene product in the test sample is greaterthan the level of the corresponding miR gene product in a controlsample. In another embodiment, the at least one miR gene product that isupregulated, and which is measured, is miR-21. In yet anotherembodiment, the pancreatic cancer is associated with metastasis and/or ahigh proliferation index. As described herein, the expression ofparticular miR gene products, which are associated with an adverseprognosis in pancreatic cancer, can prognosticate the severity of asubject's pancreatic cancer (see, e.g., Exemplification). In certainembodiments, the level of the at least one miR gene product is measuredby reverse transcribing RNA from a test sample obtained from the subjectto provide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray that comprises miRNA-specificprobe oligonucleotides to provide a hybridization profile for the testsample, and comparing the test sample hybridization profile to ahybridization profile generated from a control sample.

In one embodiment, the invention is a method of determining whether apancreatic cancer in a subject is metastatic. As described herein, mostPET-related deaths are caused by liver metastasis. Thus, identificationof metastatic pancreatic cancer can aid in determining appropriatetreatment options. In this method, the level of at least one miR geneproduct is measured in a test sample (e.g., a pancreatic cancer sample)from the subject. An alteration (e.g., an increase, a decrease) in thelevel of the miR gene product in the test sample, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof metastasis. In one embodiment, the level of the at least one miR geneproduct in the test sample is greater than the level of thecorresponding miR gene product in the control sample. In anotherembodiment, the at least one miR gene product that is upregulated ismiR-21.

In one embodiment, the invention is a method of determining whether apancreatic cancer in a subject has a high proliferation index. As isknown, pancreatic cancers having a high proliferation index have anadverse prognosis and, therefore, identification of pancreatic cancershaving a high proliferation index can also aid in determiningappropriate treatment options. In this method, the level of at least onemiR gene product is measured in a test sample (e.g., a pancreatic cancersample) from the subject. An alteration (e.g., an increase, a decrease)in the level of the miR gene product in the test sample, relative to thelevel of a corresponding miR gene product in a control sample, isindicative of a high proliferation index. In one embodiment, the levelof the at least one miR gene product in the test sample is greater thanthe level of the corresponding miR gene product in the control sample.In another embodiment, the at least one miR gene product that isupregulated is miR-21.

Identification of targets of particular miR gene products (e.g., thosemiR gene products exhibiting upregulated or downregulated expressionrelative to a control sample) can aid in elucidating mechanisms ofaction of microRNAs. As exemplified herein, particular putative targetsof select microRNAs, namely miR-103/miR-107, miR-155, miR-204/miR-211and miR-21, were identified. Analysis revealed numerous upregulated (28target genes) and downregulated (7 target genes) target genes ofparticular microRNAs in pancreatic cancer samples. As described in Table10, 28 upregulated target genes and 7 downregulated target genes ofmiR-103/miR-107 were identified in pancreatic cancer samples(Exemplification and Table 10). In addition, 2 upregulated target genesand 2 downregulated target genes of miR-103/miR-107, and 1 upregulatedtarget gene and 1 downregulated target gene of miR-21 were identified inpancreatic cancer samples (Exemplification and Table 10). Thus, in oneembodiment, expression of target genes of particular microRNAs (e.g.,those listed in Table 10) can be used to diagnose cancer (e.g.,pancreatic cancer). One of skill in the art can measure the expressionlevels of any of these target genes using known methods and/or methodsdescribed herein for measuring the expression levels of microRNAs (e.g.,quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection, microarray analysis), without undueexperimentation. In one embodiment, the target gene that is measured isProgrammed Cell Death 4 (PDCD4).

In one embodiment, the invention is a method of determining theprognosis of a subject with pancreatic cancer. In this method, the levelof PDCD4 is measured in a test sample (e.g., a pancreatic cancer sample)from the subject. An alteration (e.g., an increase, a decrease) in thelevel of PDCD4 in the test sample, relative to the level of PDCD4 in acontrol sample, is indicative of an adverse prognosis. In oneembodiment, the level of PDCD4 in the test sample is less than the levelof PDCD4 in the control sample. In another embodiment, the pancreaticcancer is associated with metastasis and/or a high proliferation index.

The level of the at least one miR gene product can be measured using avariety of techniques that are well known to those of skill in the art(e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis,solution hybridization detection). In a particular embodiment, the levelof at least one miR gene product is measured by reverse transcribing RNAfrom a test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides toone or more miRNA-specific probe oligonucleotides (e.g., a microarraythat comprises miRNA-specific probe oligonucleotides) to provide ahybridization profile for the test sample, and comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample. An alteration in the signal of at least one miRNA in thetest sample relative to the control sample is indicative of the subjecteither having, or being at risk for developing, pancreatic cancer. Inone embodiment, the signal of at least one miRNA is upregulated,relative to the signal generated from the control sample. In anotherembodiment, the signal of at least one miRNA is downregulated, relativeto the signal generated from the control sample. In a particularembodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina further embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-103-2, miR-107, miR-103-1, miR-342, miR-100, miR-24-2,miR-23a, miR-125a, miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368,miR-26b, miR-125b-2, miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2,miR-21, miR-25, miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17,miR-99b, miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197,miR-10a, miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1,miR-29b-2, miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223,miR-3p21-v, miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214,miR-7-3, miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375, miR-326, miR-155, miR-339, miR-34c, miR-345,miR-152, miR-372, miR-128a and a combination thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT at 37°C. for 40 minutes. At positions on the array where the immobilized probeDNA recognizes a complementary target cDNA in the sample, hybridizationoccurs. The labeled target cDNA marks the exact position on the arraywhere binding occurs, allowing automatic detection and quantification.The output consists of a list of hybridization events, indicating therelative abundance of specific cDNA sequences, and therefore therelative abundance of the corresponding complementary miRs, in thepatient sample. According to one embodiment, the labeled cDNA oligomeris a biotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool would allow for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a cancer (e.g.,pancreatic cancer) is quantitatively reverse transcribed to provide aset of labeled target oligodeoxynucleotides complementary to the RNA inthe sample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal). More preferably, the profile recorded includes the intensity ofthe signal from each hybridization. The profile is compared to thehybridization profile generated from a normal, e.g., noncancerous,control sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

The invention also provides methods of diagnosing whether a subject has,or is at risk for developing, a pancreatic cancer with an adverseprognosis. In this method, the level of at least one miR gene product,which is associated with an adverse prognosis in pancreatic cancer, ismeasured by reverse transcribing RNA from a test sample obtained fromthe subject to provide a set of target oligodeoxynucleotides. The targetoligodeoxynucleotides are then hybridized to one or more miRNA-specificprobe oligonucleotides (e.g., a microarray that comprises miRNA-specificprobe oligonucleotides) to provide a hybridization profile for the testsample, and the test sample hybridization profile is compared to ahybridization profile generated from a control sample. An alteration inthe signal of at least one miRNA in the test sample relative to thecontrol sample is indicative of the subject either having, or being atrisk for developing, a pancreatic cancer with an adverse prognosis. Inone embodiment, an alteration in the signal of miR-21 is indicative ofthe subject either having, or being at risk for developing, a pancreaticcancer with an adverse prognosis.

In particular embodiments of the diagnostic, prognostic and therapeuticmethods of the invention, the miR gene product is not one or more oflet7a-2, let-7c, let-7g, let-71, miR-7-2, miR-7-3, miR-9, miR-9-1,miR-10a, miR-15a, miR-15b, miR-16-1, miR-16-2, miR-17-5p, miR-20a,miR-21, miR-24-1, miR-24-2, miR-25, miR-29b-2, miR-30, miR-30a-5p,miR-30c, miR-30d, miR-31, miR-32, miR-34, miR-34a, miR-34a prec,miR-34a-1, miR-34a-2, miR-92-2, miR-96, miR-99a, miR-99b prec, miR-100,miR-103, miR-106a, miR-107, miR-123, miR-124a-1, miR-125b-1, miR-125b-2,miR-126*, miR-127, miR-128b, miR-129, miR-129-1/2 prec, miR-132,miR-135-1, miR-136, miR-137, miR-141, miR-142-as, miR-143, miR-146,miR-148, miR-149, miR-153, miR-155, miR 159-1, miR-181, miR-181b-1,miR-182, miR-186, miR-191, miR-192, miR-195, miR-196-1, miR-196-1 prec,miR-196-2, miR-199a-1, miR-199a-2, miR-199b, miR-200b, miR-202, miR-203,miR-204, miR-205, miR-210, miR-211, miR-212, miR-214, miR-215, miR-217,miR-221 and/or miR-223.

As described herein, the level of a miR gene product in a sample can bemeasured using any technique that is suitable for detecting RNAexpression levels in a biological sample. Suitable techniques (e.g.,Northern blot analysis, RT-PCR, in situ hybridization) for determiningRNA expression levels in a biological sample (e.g., cells, tissues) arewell known to those of skill in the art. In a particular embodiment, thelevel of at least one miR gene product is detected using Northern blotanalysis. For example, total cellular RNA can be purified from cells byhomogenization in the presence of nucleic acid extraction buffer,followed by centrifugation. Nucleic acids are precipitated, and DNA isremoved by treatment with DNase and precipitation. The RNA molecules arethen separated by gel electrophoresis on agarose gels according tostandard techniques, and transferred to nitrocellulose filters. The RNAis then immobilized on the filters by heating. Detection andquantification of specific RNA is accomplished using appropriatelylabeled DNA or RNA probes complementary to the RNA in question. See, forexample, Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7,the entire disclosure of which is incorporated by reference.

Suitable probes (e.g., DNA probes, RNA probes) for Northern blothybridization of a given miR gene product can be produced from thenucleic acid sequences provided in Table 1a and Table 1b and include,but are not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% complementarity to a miR gene product of interest,as well as probes that have complete complementarity to a miR geneproduct of interest. Methods for preparation of labeled DNA and RNAprobes, and the conditions for hybridization thereof to targetnucleotide sequences, are described in Molecular Cloning: A LaboratoryManual, J. Sambrook et al., eds., 2nd edition, Cold Spring HarborLaboratory Press, 1989, Chapters 10 and 11, the disclosures of which areincorporated herein by reference.

For example, the nucleic acid probe can be labeled with, e.g., aradionuclide, such as 3H, 32P, 33P, 14C, or 35S; a heavy metal; a ligandcapable of functioning as a specific binding pair member for a labeledligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; achemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing32P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare 32P-labeled nucleic acid probes with aspecific activity well in excess of 108 cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin and antibodies (e.g., anti-biotinantibodies) coupled to fluorescent dyes or enzymes that produce colorreactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique and involves depositing whole cellsonto a microscope cover slip and probing the nucleic acid content of thecell with a solution containing radioactive or otherwise labeled nucleicacid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference. Suitable probes for in situhybridization of a given miR gene product can be produced from thenucleic acid sequences provided in Table 1a and Table 1b, and include,but are not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% complementarity to a miR gene product of interest,as well as probes that have complete complementarity to a miR geneproduct of interest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods forperforming quantitative and semi-quantitative RT-PCR, and variationsthereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer (e.g., pancreatic cancer). Assessing cancer-specificexpression levels for hundreds of miR genes or gene products is timeconsuming and requires a large amount of total RNA (e.g., at least 20 μgfor each Northern blot) and autoradiographic techniques that requireradioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotide) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level inpancreatic cancer cells. As used herein, “probe oligonucleotide” or“probe oligodeoxynucleotide” refers to an oligonucleotide that iscapable of hybridizing to a target oligonucleotide. “Targetoligonucleotide” or “target oligodeoxynucleotide” refers to a moleculeto be detected (e.g., via hybridization). By “miR-specific probeoligonucleotide” or “probe oligonucleotide specific for a miR” is meanta probe oligonucleotide that has a sequence selected to hybridize to aspecific miR gene product, or to a reverse transcript of the specificmiR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from pancreatic cancer tissue,and within pancreatic cancer tissue, different prognosis states (forexample, good or poor long term survival prospects) may be determined.By comparing expression profiles of pancreatic cancer tissue indifferent states, information regarding which genes are important(including both upregulation and downregulation of genes) in each ofthese states is obtained. The identification of sequences that aredifferentially expressed in pancreatic cancer tissue or normalpancreatic tissue, as well as differential expression resulting indifferent prognostic outcomes, allows the use of this information in anumber of ways. For example, a particular treatment regime may beevaluated (e.g., to determine whether a chemotherapeutic drug acts toimprove the long-term prognosis in a particular patient). Similarly,diagnosis may be done or confirmed by comparing patient samples withknown expression profiles. Furthermore, these gene expression profiles(or individual genes) allow screening of drug candidates that suppressthe pancreatic cancer expression profile or convert a poor prognosisprofile to a better prognosis profile.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of pancreatic cancer. Therefore,altering the level of the miR gene product (e.g., by decreasing thelevel of a miR that is upregulated in pancreatic cancer cells, byincreasing the level of a miR that is downregulated in pancreatic cancercells) may successfully treat the pancreatic cancer.

Accordingly, the present invention encompasses methods of treatingpancreatic cancer in a subject, wherein at least one miR gene product isderegulated (e.g., down-regulated, upregulated) in the cells (e.g.,pancreatic cancer cells) of the subject. In one embodiment, the level ofat least one miR gene product in a test sample (e.g., a pancreaticcancer sample) is greater than the level of the corresponding miR geneproduct in a control sample. In another embodiment, the level of atleast one miR gene product in a test sample (e.g., a pancreatic cancersample is less than the level of the corresponding miR gene product in acontrol sample. When the at least one isolated miR gene product isdownregulated in the pancreatic cancer cells, the method comprisesadministering an effective amount of the at least one isolated miR geneproduct, or an isolated variant or biologically-active fragment thereof,such that proliferation of cancer cells in the subject is inhibited. Forexample, when a miR gene product is downregulated in a cancer cell in asubject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to an endogenous wild-type miR gene product (e.g., a miR geneproduct shown in Table 1a or Table 1b) that is downregulated in thecancer cell or it can be a variant or biologically-active fragmentthereof. As defined herein, a “variant” of a miR gene product refers toa miRNA that has less than 100% identity to a corresponding wild-typemiR gene product and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e.g., inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with pancreatic cancer (e.g., cell differentiation,cell growth, cell death). These variants include species variants andvariants that are the consequence of one or more mutations (e.g., asubstitution, a deletion, an insertion) in a miR gene. In certainembodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% identical to a corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated withpancreatic cancer. In certain embodiments, the biologically-activefragment is at least about 5, 7, 10, 12, 15, or 17 nucleotides inlength. In a particular embodiment, an isolated miR gene product can beadministered to a subject in combination with one or more additionalanti-cancer treatments. Suitable anti-cancer treatments include, but arenot limited to, chemotherapy, radiation therapy and combinations thereof(e.g., chemoradiation).

When the at least one isolated miR gene product is upregulated in thecancer cells, the method comprises administering to the subject aneffective amount of a compound that inhibits expression of the at leastone miR gene product, such that proliferation of pancreatic cancer cellsis inhibited. Such compounds are referred to herein as miR geneexpression-inhibition compounds. Examples of suitable miR geneexpression-inhibition compounds include, but are not limited to, thosedescribed herein (e.g., double-stranded RNA, antisense nucleic acids andenzymatic RNA molecules). In a particular embodiment, a miR geneexpression-inhibiting compound can be administered to a subject incombination with one or more additional anti-cancer treatments. Suitableanti-cancer treatments include, but are not limited to, chemotherapy,radiation therapy and combinations thereof (e.g., chemoradiation).

In a certain embodiment, the isolated miR gene product that isderegulated in pancreatic cancer (and which is administered to thesubject) is selected from the group consisting of miR-326, miR-155,miR-339, miR-34c, miR-345, miR-152, miR-372, miR-128a and a combinationthereof (or an isolated variant or biologically-active fragment of oneor more of these miRs). In a particular embodiment, the miR gene productthat is administered is not one or more of let7a-2, let-7c, let-7g,let-71, miR-7-2, miR-7-3, miR-9, miR-9-1, miR-10a, miR-15a, miR-15b,miR-16-1, miR-16-2, miR-17-5p, miR-20a, miR-21, miR-24-1, miR-24-2,miR-25, miR-29b-2, miR-30, miR-30a-5p, miR-30c, miR-30d, miR-31, miR-32,miR-34, miR-34a, miR-34a prec, miR-34a-1, miR-34a-2, miR-92-2, miR-96,miR-99a, miR-99b prec, miR-100, miR-103, miR-106a, miR-107, miR-123,miR-124a-1, miR-125b-1, miR-125b-2, miR-126*, miR-127, miR-128b,miR-129, miR-129-1/2 prec, miR-132, miR-135-1, miR-136, miR-137,miR-141, miR-142-as, miR-143, miR-146, miR-148, miR-149, miR-153,miR-155, miR 159-1, miR-181, miR-181b-1, miR-182, miR-186, miR-191,miR-192, miR-195, miR-196-1, miR-196-1 prec, miR-196-2, miR-199a-1,miR-199a-2, miR-199b, miR-200b, miR-202, miR-203, miR-204, miR-205,miR-210, miR-211, miR-212, miR-214, miR-215, miR-217, miR-221 and/ormiR-223.

As described, when the at least one isolated miR gene product isupregulated in cancer cells, the method comprises administering to thesubject an effective amount of at least one compound for inhibitingexpression of the at least one miR gene product, such that proliferationof pancreatic cancer cells is inhibited. In one embodiment, the compoundfor inhibiting expression of the at least one miR gene product inhibitsa miR gene product selected from the group consisting of miR-103-2,miR-107, miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a,miR-26a-1, miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2,miR-125b-1, miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25,miR-92-2, miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b,miR-181b-1, miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a,miR-224, miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2,miR-150, miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v,miR-128b, miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3,miR-29c, miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20,miR-129-1, miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95,miR-222, miR-30e, miR-129-2, miR-345, miR-143, miR-182, miR-1-1,miR-133a-1, miR-200c, miR-194-1, miR-210, miR-181c, miR-192, miR-220,miR-213, miR-323, miR-375 and a combination thereof.

In a related embodiment, the methods of treating pancreatic cancer in asubject additionally comprise the step of first determining the amountof at least one miR gene product in pancreatic cancer cells from thesubject, and comparing that level of the miR gene product to the levelof a corresponding miR gene product in control cells. If expression ofthe miR gene product is deregulated (e.g., downregulated, upregulated)in pancreatic cancer cells, the methods further comprise altering theamount of the at least one miR gene product expressed in the pancreaticcancer cells. In one embodiment, the amount of the miR gene productexpressed in the cancer cells is less than the amount of the miR geneproduct expressed in control cells, and an effective amount of the miRgene product, or an isolated variant or biologically-active fragmentthereof, is administered to the subject. In another embodiment, theamount of the miR gene product expressed in the cancer cells is greaterthan the amount of the miR gene product expressed in control cells, andan effective amount of at least one compound for inhibiting expressionof the at least one miR gene is administered to the subject. SuitablemiRs and compounds that inhibit expression of miR genes include, forexample, those described herein.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, pancreatic cancer, including preventing or delaying the onsetof the disease symptoms, and/or lessening the severity or frequency ofsymptoms of the disease or condition. The terms “subject”, “patient” and“individual” are defined herein to include animals, such as mammals,including, but not limited to, primates, cows, sheep, goats, horses,dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine,equine, canine, feline, rodent, or murine species. In a preferredembodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from pancreatic cancer. One skilled in the art canreadily determine an effective amount of a miR gene product to beadministered to a given subject, by taking into account factors, such asthe size and weight of the subject; the extent of disease penetration;the age, health and sex of the subject; the route of administration; andwhether the administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product that is administered to a subject can rangefrom about 5 3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in a substantially-purified form, or can exist ina cell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to the invention, the isolatedmiR gene products described herein can be used for the manufacture of amedicament for treating pancreatic cancer in a subject (e.g., a human).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, J. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in a cancer cell,using, for example, the techniques for determining miR transcript leveldiscussed herein. Inhibition can occur at the level of gene expression(i.e., by inhibiting transcription of a miR gene encoding the miR geneproduct) or at the level of processing (e.g., by inhibiting processingof a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer (e.g., pancreatic cancer). Oneskilled in the art can readily determine an effective amount of a miRexpression-inhibiting compound to be administered to a given subject, bytaking into account factors, such as the size and weight of the subject;the extent of disease penetration; the age, health and sex of thesubject; the route of administration; and whether the administration isregional or systemic.

For example, an effective amount of the expression-inhibiting compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject, as described herein.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and interfere with the expression (e.g., by inhibitingtranslation, by inducing cleavage and/or degradation) of the target miRgene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA that is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acids (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product. Nucleic acid sequences of particularhuman miR gene products are provided in Table 1a and Table 1b. Withoutwishing to be bound by any theory, it is believed that the antisensenucleic acids activate RNase H or another cellular nuclease that digeststhe miR gene product/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucleic Acids Res. 23:2092-96; Hammann etal. (1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat.No. 4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a cancer (e.g., pancreatic cancer).As used herein, to “inhibit the proliferation of a cancer cell” means tokill the cell, or permanently or temporarily arrest or slow the growthof the cell. Inhibition of cancer cell proliferation can be inferred ifthe number of such cells in the subject remains constant or decreasesafter administration of the miR gene products or miR geneexpression-inhibiting compounds. An inhibition of cancer cellproliferation can also be inferred if the absolute number of such cellsincreases, but the rate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibiting compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression-inhibiting compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds. Inone embodiment, the cells are transfected with a plasmid or viral vectorcomprising sequences encoding at least one miR gene product or miR geneexpression-inhibiting compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/105 cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 105 cells can be used.

A miR gene product or miR gene expression-inhibiting compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibiting compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene expression-inhibitingcompound. Suitable delivery reagents include, e.g., the Mirus TransitTKO lipophilic reagent; LIPOFECTIN; lipofectamine; cellfectin;polycations (e.g., polylysine) and liposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibiting compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibiting compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) or derivatives thereof; e.g.,methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers, such aspolyacrylamide or poly N-vinyl pyrrolidone; linear, branched, ordendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalcohol and polyxylitol to which carboxylic or amino groups arechemically linked, as well as gangliosides, such as ganglioside GM1.Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,are also suitable. In addition, the opsonization-inhibiting polymer canbe a block copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating pancreatic cancer. In oneembodiment, the pharmaceutical composition comprises at least oneisolated miR gene product, or an isolated variant or biologically-activefragment thereof, and a pharmaceutically-acceptable carrier. In aparticular embodiment, the at least one miR gene product corresponds toa miR gene product that has a decreased level of expression inpancreatic cancer cells relative to suitable control cells (i.e., it isdownregulated). In a certain embodiment, the isolated miR gene productis selected from the group consisting of miR-326, miR-155, miR-339,miR-34c, miR-345, miR-152, miR-372, miR-128a and a combination thereof.In one embodiment, the isolated miR gene product is not miR-15a ormiR-16-1. In an additional embodiment, the miR gene product is notmiR-210 or miR-212. In another embodiment, the miR gene product is notmiR-21, miR-143, miR-205 or miR-9. In yet another embodiment, the miRgene product is not miR-21, miR-191, miR-126*, miR-210, miR-155,miR-143, miR-205, miR-126, miR-30a-5p, miR-140, miR-214, miR-218-2,miR-145, miR-106a, miR-192, miR-203, miR-150, miR-220, miR-212 or miR-9.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound and apharmaceutically-acceptable carrier. In a particular embodiment, the atleast one miR expression-inhibition compound is specific for a miR geneproduct whose expression is greater in pancreatic cancer cells thancontrol cells (i.e., it is upregulated). In certain embodiments, the miRexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-103-2, miR-107,miR-103-1, miR-342, miR-100, miR-24-2, miR-23a, miR-125a, miR-26a-1,miR-24-1, miR-191, miR-15a, miR-368, miR-26b, miR-125b-2, miR-125b-1,miR-26a-2, miR-335, miR-126, miR-1-2, miR-21, miR-25, miR-92-2,miR-130a, miR-93, miR-16-1, miR-145, miR-17, miR-99b, miR-181b-1,miR-146, miR-181b-2, miR-16-2, miR-99a, miR-197, miR-10a, miR-224,miR-92-1, miR-27a, miR-221, miR-320, miR-7-1, miR-29b-2, miR-150,miR-30d, miR-29a, miR-23b, miR-135a-2, miR-223, miR-3p21-v, miR-128b,miR-30b, miR-29b-1, miR-106b, miR-132, miR-214, miR-7-3, miR-29c,miR-367, miR-30c-2, miR-27b, miR-140, miR-10b, miR-20, miR-129-1,miR-340, miR-30a, miR-30c-1, miR-106a, miR-32, miR-95, miR-222, miR-30e,miR-129-2, miR-345, miR-143, miR-182, miR-1-1, miR-133a-1, miR-200c,miR-194-1, miR-210, miR-181c, miR-192, miR-220, miR-213, miR-323,miR-375 and a combination thereof. In one embodiment, the isolated miRgene product is not specific for miR-15a or miR-16-1. In anotherembodiment, the miR gene product is not specific for miR-210 or miR-212.In yet another embodiment, the miR gene product is not specific formiR-21, miR-143, miR-205 or miR-9. In still another embodiment, the miRgene product is not specific for miR-21, miR-191, miR-126*, miR-210,miR-155, miR-143, miR-205, miR-126, miR-30a-5p, miR-140, miR-214,miR-218-2, miR-145, miR-106a, miR-192, miR-203, miR-150, miR-220,miR-212 or miR-9.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example, as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising a sequence encoding the miR gene product or miRgene expression-inhibition compound) (e.g., 0.1 to 90% by weight), or aphysiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical composition of the invention additionally comprises oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising a sequence encoding the miR geneproduct or miR gene expression-inhibition compound), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is not miR-15, miR-16, miR-143 and/or miR-145.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisinga sequence encoding the miR gene product or miR geneexpression-inhibition compound) that is resistant to degradation bynucleases. One skilled in the art can readily synthesize nucleic acidsthat are nuclease resistant, for example by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising a sequence encoding the miR gene product or miR geneexpression-inhibition compound) encapsulated in a liposome as describedabove, and a propellant. A carrier can also be included as desired;e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisinga sequence encoding the miR gene product or miR geneexpression-inhibition compound) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel and FOLFOX4.

The invention also encompasses methods of identifying an anti-pancreaticcancer agent, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in pancreatic cancer cells. An increase inthe level of the miR gene product in the cell, relative to a to asuitable control (e.g., the level of the miR gene product in a controlcell), is indicative of the test agent being an anti-pancreatic canceragent. In a particular embodiment, the at least one miR gene productassociated with decreased expression levels in pancreatic cancer cellsis selected from the group consisting of miR-326, miR-155, miR-339,miR-34c, miR-345, miR-152, miR-372, miR-128a and a combination thereof.In one embodiment, the miR gene product is not one or more of let7a-2,let-7c, let-7g, let-71, miR-7-2, miR-7-3, miR-9, miR-9-1, miR-10a,miR-15a, miR-15b, miR-16-1, miR-16-2, miR-17-5p, miR-20a, miR-21,miR-24-1, miR-24-2, miR-25, miR-29b-2, miR-30, miR-30a-5p, miR-30c,miR-30d, miR-31, miR-32, miR-34, miR-34a, miR-34a prec, miR-34a-1,miR-34a-2, miR-92-2, miR-96, miR-99a, miR-99b prec, miR-100, miR-103,miR-106a, miR-107, miR-123, miR-124a-1, miR-125b-1, miR-125b-2,miR-126*, miR-127, miR-128b, miR-129, miR-129-1/2 prec, miR-132,miR-135-1, miR-136, miR-137, miR-141, miR-142-as, miR-143, miR-146,miR-148, miR-149, miR-153, miR-155, miR 159-1, miR-181, miR-181b-1,miR-182, miR-186, miR-191, miR-192, miR-195, miR-196-1, miR-196-1 prec,miR-196-2, miR-199a-1, miR-199a-2, miR-199b, miR-200b, miR-202, miR-203,miR-204, miR-205, miR-210, miR-211, miR-212, miR-214, miR-215, miR-217,miR-221 and/or miR-223.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in pancreatic cancer cells. A decreasein the level of the miR gene product associated with increasedexpression levels in pancreatic cancer in the cell, relative to asuitable control (e.g., the level of the miR gene product in a controlcell), is indicative of the test agent being an anti-pancreatic canceragent. In a particular embodiment, the at least one miR gene productassociated with increased expression levels in pancreatic cancer cellsis selected from the group consisting of miR-103-2, miR-107, miR-103-1,miR-342, miR-100, miR-24-2, miR-23a, miR-125a, miR-26a-1, miR-24-1,miR-191, miR-15a, miR-368, miR-26b, miR-125b-2, miR-125b-1, miR-26a-2,miR-335, miR-126, miR-1-2, miR-21, miR-25, miR-92-2, miR-130a, miR-93,miR-16-1, miR-145, miR-17, miR-99b, miR-181b-1, miR-146, miR-181b-2,miR-16-2, miR-99a, miR-197, miR-10a, miR-224, miR-92-1, miR-27a,miR-221, miR-320, miR-7-1, miR-29b-2, miR-150, miR-30d, miR-29a,miR-23b, miR-135a-2, miR-223, miR-3p21-v, miR-128b, miR-30b, miR-29b-1,miR-106b, miR-132, miR-214, miR-7-3, miR-29c, miR-367, miR-30c-2,miR-27b, miR-140, miR-10b, miR-20, miR-129-1, miR-340, miR-30a,miR-30c-1, miR-106a, miR-32, miR-95, miR-222, miR-30e, miR-129-2,miR-345, miR-143, miR-182, miR-1-1, miR-133a-1, miR-200c, miR-194-1,miR-210, miR-181c, miR-192, miR-220, miR-213, miR-323, miR-375 and acombination thereof. In one embodiment, the miR gene product is not oneor more of let7a-2, let-7c, let-7g, let-71, miR-7-2, miR-7-3, miR-9,miR-9-1, miR-10a, miR-15a, miR-15b, miR-16-1, miR-16-2, miR-17-5p,miR-20a, miR-21, miR-24-1, miR-24-2, miR-25, miR-29b-2, miR-30,miR-30a-5p, miR-30c, miR-30d, miR-31, miR-32, miR-34, miR-34a, miR-34aprec, miR-34a-1, miR-34a-2, miR-92-2, miR-96, miR-99a, miR-99b prec,miR-100, miR-103, miR-106a, miR-107, miR-123, miR-124a-1, miR-125b-1,miR-125b-2, miR-126*, miR-127, miR-128b, miR-129, miR-129-1/2 prec,miR-132, miR-135-1, miR-136, miR-137, miR-141, miR-142-as, miR-143,miR-146, miR-148, miR-149, miR-153, miR-155, miR 159-1, miR-181,miR-181b-1, miR-182, miR-186, miR-191, miR-192, miR-195, miR-196-1,miR-196-1 prec, miR-196-2, miR-199a-1, miR-199a-2, miR-199b, miR-200b,miR-202, miR-203, miR-204, miR-205, miR-210, miR-211, miR-212, miR-214,miR-215, miR-217, miR-221 and/or miR-223.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described herein.

The invention will now be illustrated by the following non-limitingexamples.

Exemplification

Materials and Methods

Patient Data, Neoplastic-Cell Enrichment and RNA Extraction.

The clinicopathological characteristics of 40 PET and four PACC,retrieved from the frozen tissue bank of the Pathology Department of theUniversity of Verona, Italy, are reported in Table 2. All tumors weresporadic, as assessed by personal and family histories obtained bydirect interview of patients. PET were diagnosed by histopathologic andcell marker analysis, and classified according to WHO criteria (Kloppel,G., et al., “The Gastroenteropancreatic Neuroendocrine Cell System andIts Tumors: The WHO Classification.” Ann. N.Y. Acad. Sci. 1014:13-27(2004)). They included 28 nonfunctional and 12 functional tumors. The 28NF-PET included 11 well-differentiated endocrine tumors (WDET) and 18well differentiated endocrine carcinomas (WDEC). The 12 F-PET wereinsulinomas, comprising 11 WDET and 1 WDEC. WDET were considered witheither benign or uncertain biological behavior in accordance with theWHO criteria, that considers tumor size, Ki-67 proliferation index andvascular invasion (Table 2). Diagnosis of PACC was confirmed byimmunohistochemical expression of lipase, amylase and trypsin inneoplastic cells. As a control, normal pancreas was taken in 12corresponding patient specimens.

A neoplastic cellularity of more than 90% was obtained in all cases bymicrodissection or cryostat enrichment. Total RNA was extracted withTrizol (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions from at least ten 20-30 μm thick cryostat sections,checking the cell composition of the sample every five sections. Theintegrity of total RNA was confirmed in each case using the Agilent 2100Bioanalyzer (Agilent Technologies, Palo Alto, Calif.).

TABLE 2 Clinicopathological data of the pancreatic endocrine and acinartumors. Metastases Vascular Insulind Ki67e Casea Sex Age Size (cm)Diagnosisb Invasionc LNf Liver Invasion IHC (%) F- K29 F 46 9 WDEC yesyes yes yes pos-w 20 F- K11 F 46 1.5 WDET-b no no no no pos 2 F- K36 M23 1.3 WDET-b no no no no pos-w 2 F- Q2 M 51 1.5 WDET-b no no no no pos2 F- Q4 F 63 1.2 WDET-b no no no no pos-w 1 F- Q6 M 51 1.3 WDET-b no nono no pos 1 F- K20 F 27 5 WDET-u no no no no pos 5 F- K47 M 66 2.5WDET-u no no no no pos-w 1 F- K66 M 33 5 WDET-u no no no yes pos-w 10 F-K69 M 67 2.5 WDET-u no no no no pos n.a. g F- K80 F 35 4 WDET-u no no nono pos 1 F- Q14 M 41 1.3 WDET-u no no no no pos 3 NF- K14 M 48 11 WDECyes no no yes neg 5 NF- K15 M 44 18 WDEC yes yes no yes neg 20 NF- K16 M48 6.5 WDEC yes yes no yes n.a. 3 NF- K19 F 37 4 WDEC no yes no no neg30 NF- K23 M 56 7 WDEC yes yes yes yes pos 5 NF- K25 F 60 2 WDEC yes nono yes neg 2 NF- K3 M 42 4.5 WDEC yes yes yes yes neg 8 NF- K31 F 54 3WDEC yes yes yes yes neg 20 NF- K32 F 61 4.5 WDEC no no yes yes neg 15NF- K37 F 35 3 WDEC no yes no no neg 2 NF- K42 F 65 4.5 WDEC yes yes nono neg 2 NF- K43 M 53 4 WDEC yes yes yes yes pos-w 10 NF- K6 F 51 12WDEC no no yes yes neg 7 NF- K76 F 40 5.5 WDEC no yes no yes neg 5 NF-K9 F 70 5.5 WDEC yes yes yes yes n.a. 25 NF- Q12 M 56 4.5 WDEC yes no noyes neg 2 NF- Q5 M 38 3 WDEC yes yes yes yes neg 3 NF- K63 M 58 1.2WDET-b no no no no neg 2 NF- K8 F 42 1.5 WDET-b no no no no neg 2 NF-K10 F 66 1.5 WDET-u no no no no neg 2 NF- K13 F 66 2 WDET-u no no no noneg 2 NF- K2 M 68 1.5 WDET-u no no no yes n.a. 1 NF- K24 F 49 11 WDET-uno no no no neg 1 NF- K35 M 40 8 WDET-u no no no no neg 2 NF- K41 M 39 3WDET-u no no no yes neg 1 NF- K7 M 57 2 WDET-u no no no no neg 3 NF- K75F 69 2.5 WDET-u no no no no neg 1 NF- Q13 F 65 3 WDET-u no no no yes neg2 AC- K53 M 36 7 ACC no no no yes neg 10 AC- K54 M 64 5 ACC no no no noneg 12 AC- K58 M 39 6 ACC yes yes no yes neg 15 AC- K60 M 52 17 ACC yesyes no yes neg n.a.

aCases are identified by a random assigned number precede by F or NF ifthey are functioning or nonfunctioning endocrine tumors, respectively.

bWDEC, well differentiated endocrine carcinoma; WDET-b, welldifferentiated endocrine tumor with benign behavior; WDET-u, welldifferentiated endocrine tumor with uncertain biological behavior.

cInvasion of peripancreatic fat and/or adjacent organs (e.g. duodenum,choledocus, spleen).

dIHC, immunohistochemistry; pos, positive; pos-w, positive with weaksignal; neg, negative.

eProliferation index measured by Ki67 immunohistochemistry.

fLN, lymph nodes.

gn.a., not available.

MicroRNA Microarray Hybridization and Quantification.

MicroRNA labeling and hybridization on microRNA microarray chips wereperformed as previously described (Liu, C. G., et al., “AnOligonucleotide Microchip for Genome-Wide microRNA Profiling in Humanand Mouse Tissues.” Proc. Natl. Acad. Sci. USA 101:9740-44 (2004)).Briefly, 5 μg of total RNA from each sample was reverse transcribedusing biotin end-labeled random octamers. Hybridization was carried outon our custom microRNA microarray chip (OSU-CCC version 2.0), whichcontains probes for 460 mature microRNAs (235 Homo sapiens, 222 Musmusculus, and 3 Arabidopsis thaliana) spotted in quadruplicate withannotated active sites. Often, more than one probe set exists for agiven mature microRNA. Additionally, there are quadruplicate probescorresponding to most pre-microRNA for detecting the microRNA precursor.The microarray also includes probes for several splicing snRNAs,including U6. Hybridization signals were detected withStreptavidin-Alexa647 conjugate and scanned using Axon 4000B. Scanimages were quantified using the Genepix 6.0 software (Axon Instruments(now Molecular Devices Corp.), Sunnyvale, Calif.).

Computational Analyses of microRNA Microarray Data.

Most of the analysis and graphics were generated using R software v.2.0.1 and Bioconductor v. 1.6 packages (Gentleman, R. C., et al.,“Bioconductor: Open Software Development for Computational Biology andBioinformatics.” Genome Biol. 5:R80 (2004)). Sequentially, the blank andprobe controls spots were removed from the dataset of the 56 microRNAmicroarrays, and the local background was then subtracted from themedian signal. Next, the data was normalized using avariance-stabilizing transformation stratified, within each array, bygrid in the vsn package. Subsequently, genefilter package was used toremove all the spots whose intensities were lower than 99th percentileof the blank spots in all the arrays. The relative hybridization toblank/negative control probes and subsequent Northern analysis indicatedthat the absolute value of log-transformed signals less than 4.5 areunreliable.

The data obtained were further analyzed by direct two class unpairedcomparison using the same package. The tables of differentiallyexpressed microRNAs were obtained applying the input criteria (based onfold-change and delta value) that are specifically reported in theirtitle. In order to increase stringency, microRNA probes were furtherfiltered retaining those that had at least three significant replicas.

Hierarchical cluster analysis was performed using the aggregate valuesof replicate spots obtained applying Tukey's median polish algorithm.The analysis was done using the first 200 probes with the highestinterquartile range, which contained the mature microRNA sequences. Thedistance metrics used to cluster samples and genes were Pearsoncorrelation and Euclidean distance, respectively. The agglomerativemethod was the complete-linkage. The output was visualized using MapleTree (version 0.2.3.2) (www.mapletree.sourceforge.net). All data weresubmitted using MIAMExpress to the Array Express database.

The level of coordinate expression between microRNAs was measured byPearson correlation and microRNA genes were assigned to the same clusterwhen their distance was below 50 kb (Baskerville, S, and D. P. Bartel,“Microarray Profiling of microRNAs Reveals Frequent Coexpression withNeighboring miRNAs and Host Genes.” RNA 11: 241-47 (2005)). Next, theset of correlation values measured between microRNAs belonging to thesame cluster were compared to the set of correlation values measuredbetween each microRNA in a cluster vs. all other microRNAs out of thatcluster using Mann-Whitney non-parametric test.

Northern Blotting.

Five μg of total RNAs were run on 15% Criterion precast PAGE/Urea gels(Bio-Rad, Hercules, Calif.), transferred onto Hybond-N+ membrane(Amersham Biosciences, Piscataway, N.J.) and hybridized overnight with32P end-labeled DNA probes at 37° C. in ULTRAhyb™-Oligo hybridizationbuffer (Ambion, Austin, Tex.). Membranes were washed at 37° C. twice for30 minutes each with 2×SSC/0.5% SDS. The DNA probes were antisenseoligonucleotides relative to the mature microRNAs and to 5S RNA as acontrol. Filters were analyzed using a Typhoon 9410 phoshorimager(Amersham Biosciences, Piscataway, N.J.) and quantified using ImageQuantTL (Amersham Biosciences, Piscataway, N.J.). Blots were stripped byboiling in 0.1% aqueous SDS for five minutes and were reprobed severaltimes.

Results

MicroRNA expression profiles were determined for 12 normal pancreassamples and 44 pancreatic tumors, including 40 PETs and four PACCs,using a custom microarray. This platform was proved to give robustresults, as validated by several previous studies (Liu, C. G., et al.,Proc. Natl. Acad. Sci. USA 101:9740-44 (2004); Calin, G., et al., NewEngl. J. Med. 353(17):1793-1801 (2005); Iorio, M. V., et al., CancerRes. 65:7065-70 (2005)). Further support was provided by the findingthat microRNAs that are physically linked in genomic clusters werecoexpressed, confirming that grouped microRNA genes show coordinateexpression (Baskerville, S., and D. P. Bartel, RNA 11:241-47 (2005);Altuvia, Y., et al., Nucleic Acids Res. 33:2697-2706 (2005)).

The unsupervised analysis by hierarchical clustering, using the twohundred most variable microRNAs, showed a common microRNA expressionpattern distinguishing pancreatic endocrine and acinar tumors fromnormal pancreas (FIGS. 1A-1E). Notably, PACCs fell into a unique clusterthat was part of the wider cluster including all PETs, while there wasno distinctive pattern between insulinomas and NF-PET.

Class comparison analysis confirmed the differential expression ofseveral microRNAs between PACC or PET and normal tissue, while a smallernumber of microRNAs were differentially expressed between PET and PACC,as well as between the WDEC subgroup of PET and PACC. In particular, PETshowed 87 upregulated and 8 downregulated microRNAs, as compared tonormal pancreas (Table 3), while PACC had 30 microRNAs upregulated and 7downregulated (Table 4). Only ten microRNAs were differentiallyexpressed between PET and PACC (Table 5), and four were unique to WDEC,with respect to PACC (Table 6).

TABLE 3 Differentially-expressed microRNAs between PETs and Normal BulkPancreas (FDR: 0% on the 90th percentile and 2-Fold). Fold q-valueActive microRNA MicroRNA Name Score (a) Change (b) Accession IDChromosome site (c) Families (d) Upregulated microRNAs hsa-mir-103-212.017 14.52 0 MI0000108 20p13 Yes 20 hsa-mir-107 11.885 15.94 0MI0000114 10q23.31 Yes 20 hsa-mir-103-1 11.296 14.18 0 MI0000109 5q34Yes 20 hsa-mir-342 10.970 10.09 0 MI0000805 14q32.2 Yes 186 hsa-mir-10010.277 9.71 0 MI0000102 11q24.1 Yes 17 hsa-mir-24-2 10.116 6.20 0MI0000081 19p13.12 Yes 38 hsa-mir-23a 9.468 7.11 0 MI0000079 19p13.12Yes 32 hsa-mir-125a 9.011 7.52 0 MI0000469 19q13.41 Yes 9 hsa-mir-26a-18.787 5.34 0 MI0000083 3p22.3 Yes 39 hsa-mir-24-1 8.762 4.67 0 MI00000809q22.32 Yes 38 hsa-mir-191 8.570 5.78 0 MI0000465 3p21.31 Yes 176hsa-mir-15a 7.774 3.94 0 MI0000069 13q14.2 Yes 24 hsa-mir-368 7.718 6.610 MI0000776 14q32.31 Yes 124 hsa-mir-26b 7.710 5.15 0 MI0000084 2q35 Yes39 hsa-mir-125b-2 7.687 6.52 0 MI0000470 21q21.1 Yes 9 hsa-mir-125b-17.623 8.08 0 MI0000446 11q24.1 Yes 9 hsa-mir-26a-2 7.498 5.52 0MI0000750 12q14.1 Yes 39 hsa-mir-335 7.361 2.94 0 MI0000816 7q32.2 Yes192 hsa-mir-126 7.210 6.06 0 MI0000471 9q34.3 Yes 127 hsa-mir-1-2 7.1706.58 0 MI0000437 18q11.2 Yes 35 hsa-mir-21 7.030 5.78 0 MI000007717q23.2 Yes 61 hsa-mir-25 7.017 4.16 0 MI0000082 7q22.1 Yes 69hsa-mir-92-2 7.005 3.86 0 MI0000094 Xq26.2 Yes 30 hsa-mir-130a 6.9854.32 0 MI0000448 11q12.1 Yes 50 hsa-mir-93 6.971 3.56 0 MI0000095 7q22.1Yes 2 hsa-mir-16-1 6.785 4.57 0 MI0000070 13q14.2 Yes 46 hsa-mir-1456.770 4.49 0 MI0000461 5q32 Yes 70 hsa-mir-17 6.759 4.03 0 MI000007113q31.3 Yes 2 hsa-mir-99b 6.681 5.91 0 MI0000746 19q13.41 Yes 17hsa-mir-181b-1 6.645 4.80 0 MI0000270 1q31.3 Yes 44 hsa-mir-146 6.6394.29 0 MI0000477 5q33.3 Yes 109 hsa-mir-181b-2 6.613 4.45 0 MI00006839q33.3 Yes 44 hsa-mir-16-2 6.613 3.90 0 MI0000115 3q25.33 Yes 46hsa-mir-99a 6.561 4.35 0 MI0000101 21q21.1 Yes 17 hsa-mir-197 6.512 2.440 MI0000239 1p13.3 Yes 112 hsa-mir-10a 6.447 4.44 0 MI0000266 17q21.32Yes 33 hsa-mir-224 6.445 2.93 0 MI0000301 Xq28 Yes 85 hsa-mir-92-1 6.4423.08 0 MI0000093 13q31.3 Yes 30 hsa-mir-27a 6.255 3.34 0 MI000008519p13.12 Yes 40 hsa-mir-221 6.171 8.97 0 MI0000298 Xp11.3 Yes 90hsa-mir-320 6.143 2.38 0 MI0000542 8p21.3 Yes 162 hsa-mir-7-1 6.133 4.840 MI0000263 9q21.32 Yes 12 hsa-mir-29b-2 6.110 4.07 0 MI0000107 1q32.2Yes 8 hsa-mir-150 6.033 2.63 0 MI0000479 19q13.33 Yes 178 hsa-mir-30d5.930 5.11 0 MI0000255 8q24.22 Yes 28 hsa-mir-29a 5.930 3.87 0 MI00000877q32.3 Yes 8 hsa-mir-23b 5.803 3.02 0 MI0000439 9q22.32 Yes 32hsa-mir-135a-2 5.675 2.86 0 MI0000453 12q23.1 Yes 31 hsa-mir-223 5.5803.46 0 MI0000300 Xq12 Yes 68 hsa-mir-3p21-v 5.579 2.32 0 NA NA Yes NAhsa-mir-128b 5.557 4.35 0 MI0000727 3p22.3 Yes 51 hsa-mir-30b 5.551 4.250 MI0000441 8q24.22 Yes 27 hsa-mir-29b-1 5.456 3.14 0 MI0000105 7q32.3Yes 8 hsa-mir-106b 5.448 2.37 0 MI0000734 7q22.1 Yes 2 hsa-mir-132 5.4456.39 0 MI0000449 17p13.3 Yes 110 hsa-mir-214 5.440 2.58 0 MI00002901q24.3 Yes 62 hsa-mir-7-3 5.418 4.72 0 MI0000265 19p13.3 Yes 12hsa-mir-29c 5.406 3.12 0 MI0000735 1q32.2 Yes 8 hsa-mir-367 5.398 3.47 0MI0000775 4q25 Yes NA hsa-mir-30c-2 5.356 4.10 0 MI0000254 6q13 Yes 27hsa-mir-27b 5.344 2.98 0 MI0000440 9q22.32 Yes 40 hsa-mir-140 5.251 3.090 MI0000456 16q22.1 Yes 95 hsa-mir-10b 5.218 3.46 0 MI0000267 2q31.1 Yes33 hsa-mir-20 5.208 3.45 0 MI0000076 13q31.3 Yes 2 hsa-mir-129-1 5.1433.97 0 MI0000252 7q32.1 No 93 hsa-mir-340 5.123 2.67 0 MI0000802 5q35.3Yes 181 hsa-mir-30a 5.119 3.29 0 MI0000088 6q13 Yes 28 hsa-mir-30c-15.065 3.88 0 MI0000736 1p34.2 Yes 27 hsa-mir-106a 4.974 2.81 0 MI0000113Xq26.2 Yes 2 hsa-mir-32 4.763 2.34 0 MI0000090 9q31.3 Yes 63 hsa-mir-954.582 2.53 0 MI0000097 4p16.1 Yes 87 hsa-mir-222 4.417 3.48 0 MI0000299Xp11.3 Yes 103 hsa-mir-30e 4.149 4.01 0 MI0000749 1p34.2 Yes 28hsa-mir-129-2 3.946 2.27 0 MI0000473 11p11.2 Yes 93 hsa-mir-345 3.9092.31 0 MI0000825 14q32.2 Yes 193 hsa-mir-143 3.808 2.58 0 MI0000459 5q32Yes 74 hsa-mir-182 3.762 3.78 0 MI0000272 7q32.2 Yes 126 hsa-mir-1-13.674 2.22 0 MI0000651 20q13.33 Yes 35 hsa-mir-133a-1 3.583 2.66 0MI0000450 18q11.2 Yes 25 hsa-mir-200c 3.463 3.08 0 MI0000650 12p13.31Yes 111 hsa-mir-194-1 3.345 3.57 0 MI0000488 1q41 Yes 54 hsa-mir-2103.330 2.73 0 MI0000286 11p15.5 Yes 134 hsa-mir-181c 3.116 2.38 0MI0000271 19p13.12 Yes 21 hsa-mir-192 2.905 2.71 0 MI0000234 11q13.1 Yes64 hsa-mir-220 2.877 2.45 0 MI0000297 Xq25 Yes 101 hsa-mir-213 2.8252.61 0 MI0000289 1q31.3 Yes 21 hsa-mir-323 2.589 3.75 0 MI000080714q32.31 Yes 23 Downregulated microRNAs hsa-mir-326 −6.697 0.36 0MI0000808 11q13.4 Yes 148 hsa-mir-155 −6.357 0.21 0 MI0000681 21p21.3Yes 150 hsa-mir-339 −5.531 0.41 0 MI0000815 7q22.3 Yes 191 hsa-mir-34c−4.924 0.42 0 MI0000743 11q23.1 Yes 94 hsa-mir-345 −4.873 0.49 0MI0000825 14q32.2 Yes 193 hsa-mir-152 −4.837 0.50 0 MI0000462 17q21.32No 59 hsa-mir-372 −4.221 0.43 0 MI0000780 19q13.42 Yes 217 hsa-mir-128a−4.149 0.50 0 MI0000447 2q21.3 No 51 Score: T-statistic values. q value:this is the lowest False Discovery Rate at which the gene is calledsignificant. It is like the familiar “p-value”, adapted to the analysisof a large number of genes. Active site: indicates if the probe containsthe mature form of the microRNA. (d) microRNA Family: related microRNAbased on homology in the hairpin flanking reions as reported in ThemiRBase Sequence Database - Release 7.0.

TABLE 4 Differentially-expressed microRNAs between ACCss and Normal BulkPancreas (FDR: 0% on the 90th percentile and 2-Fold). q-value Activesite microRNA MicroRNA Name Score (a) Fold Change (b) Accession IDChromosome (c) Families (d) Upregulated microRNAs hsa-mir-103-2 3.9266.46 0 MI0000108 20p13 Yes 20 hsa-mir-25 3.871 4.79 0 MI0000082 7q22.1Yes 69 hsa-mir-200c 3.828 3.88 0 MI0000650 12p13.31 Yes 111 hsa-mir-3353.702 3.46 0 MI0000816 7q32.2 Yes 192 hsa-mir-21 3.532 5.22 0 MI000007717q23.2 Yes 61 hsa-mir-103-1 3.474 6.09 0 MI0000109 5q34 Yes 20hsa-mir-92-1 3.419 3.13 0 MI0000093 13q31.3 Yes 30 hsa-mir-181b-2 3.3693.35 0 MI0000683 9q33.3 Yes 44 hsa-mir-191 3.344 4.95 0 MI00004653p21.31 Yes 176 hsa-mir-93 3.299 3.60 0 MI0000095 7q22.1 Yes 2hsa-mir-26a-1 3.248 3.85 0 MI0000083 3p22.3 Yes 39 hsa-mir-17 3.211 3.760 MI0000071 13q31.3 Yes 2 hsa-mir-20 3.201 3.37 0 MI0000076 13q31.3 Yes2 hsa-mir-107 3.195 6.16 0 MI0000114 10q23.31 Yes 20 hsa-mir-26b 3.1854.15 0 MI0000084 2q35 Yes 39 hsa-mir-215 3.123 4.70 0 MI0000291 1q41 Yes64 hsa-mir-92-2 3.088 3.60 0 MI0000094 Xq26.2 Yes 30 hsa-mir-192 3.0443.24 0 MI0000234 11q13.1 Yes 64 hsa-mir-342 2.997 3.37 0 MI000080514q32.2 Yes 186 hsa-mir-100 2.918 3.36 0 MI0000102 11q24.1 Yes 17hsa-mir-3p21-v 2.895 2.35 0 NA NA Yes NA hsa-mir-106a 2.833 3.02 0MI0000113 Xq26.2 Yes 2 hsa-mir-15a 2.809 2.83 0 MI0000069 13q14.2 Yes 24hsa-mir-23a 2.748 4.23 0 MI0000079 19p13.12 Yes 32 hsa-mir-181b-1 2.7324.00 0 MI0000270 1q31.3 Yes 44 hsa-mir-128b 2.709 2.58 0 MI00007273p22.3 Yes 51 hsa-mir-106b 2.485 2.50 0 MI0000734 7q22.1 Yes 2hsa-mir-194-1 2.432 3.21 0 MI0000488 1q41 Yes 54 hsa-mir-219-1 2.4042.06 0 MI0000296 6q21.32 Yes 47 hsa-mir-24-2 2.388 3.27 0 MI000008119p13.12 Yes 38 Downregulated microRNAs hsa-mir-218-2 −4.346 0.27 0MI0000295 5q34 Yes 29 hsa-mir-339 −4.272 0.27 0 MI0000815 7q22.3 Yes 191hsa-mir-326 −4.037 0.26 0 MI0000808 11q13.4 Yes 148 hsa-mir-34c −3.5250.27 0 MI0000743 11q23.1 Yes 94 hsa-mir-152 −3.507 0.31 0 MI000046217q21.32 No 59 hsa-mir-138-2 −3.398 0.33 0 MI0000455 16q13 Yes 88hsa-mir-128a −3.021 0.33 0 MI0000447 2q21.3 No 51 Score: T-statisticvalues. q value: this is the lowest False Discovery Rate at which thegene is called significant. It is like the familiar “p-value”, adaptedto the analysis of a large number of genes. Active site: indicates ifthe probe contains the mature form of the microRNA. (d) microRNA Family:related microRNA based on homology in the hairpin flanking reions asreported in The miRBase Sequence Database - Release 7.0.

TABLE 5 Differentially-expressed microRNAs between PETs and PACC (FDR:1% on the 90th percentile and 2-fold). Upregulated microRNAs Foldq-value Active microRNA MicroRNA Name Score (a) Change (b) Accession IDChromosome site (c) Families (d) hsa-mir-125a 4.382 4.19 0 MI000046919q13.41 Yes 9 hsa-mir-99a 3.711 3.62 0 MI0000101 21q21.1 Yes 17hsa-mir-99b 3.287 4.25 0 MI0000746 19q13.41 Yes 17 hsa-mir-125b-1 3.2713.33 0 MI0000446 11q24.1 Yes 9 hsa-mir-342 3.152 3.00 0 MI000080514q32.2 Yes 186 hsa-mir-130a 3.101 2.69 0 MI0000448 11q12.1 Yes 50hsa-mir-100 3.028 2.93 0 MI0000102 11q24.1 Yes 17 hsa-mir-132 2.952 5.440 MI0000449 17p13.3 Yes 110 hsa-mir-129-2 2.910 3.86 0 MI0000473 11p11.2Yes 93 hsa-mir-125b-2 2.886 2.94 0 MI0000470 21q21.1 Yes 9 Score:T-statistic values. q value: this is the lowest False Discovery Rate atwhich the gene is called significant. It is like the familiar “p-value”,adapted to the analysis of a large number of genes. Active site:indicates if the probe contains the mature form of the microRNA. (d)microRNA Family: related microRNA based on homology in the hairpinflanking reions as reported in The miRBase Sequence Database - Release7.0.

TABLE 6 Differentially-expressed microRNAs between WDEC and ACC (FDR: 1%on the 90th percentile and 2-fold). q-value Active site microRNAMicroRNA Name Score (a) Fold Change (b) Accession ID Chromosome (c)Families (d) Upregulated microRNAs hsa-mir-125a 3.785 3.76 0 MI000046919q13.41 Yes 9 hsa-mir-99a 3.186 3.65 0 MI0000101 21q21.1 Yes 17hsa-mir-132 2.969 4.84 0 MI0000449 17p13.3 Yes 110 DownregulatedmicroRNAs hsa-mir-148a −3.781 0.21 0 MI0000253 7p15.2 Yes 59 Score:T-statistic values. q value: this is the lowest False Discovery Rate atwhich the gene is called significant. It is like the familiar “p-value”,adapted to the analysis of a large number of genes. Active site:indicates if the probe contains the mature form of the microRNA. (d)microRNA Family: related microRNA based on homology in the hairpinflanking reions as reported in The miRBase Sequence Database - Release7.0.

TABLE 7 Differentially-expressed microRNAs between Insulomas andNon-Functioning PETs (FDR: 1% on the 90th percentile and 2-fold).Upregulated microRNAs Fold Active site microRNA MicroRNA Name Score (a)Change q-value (b) Accession ID Chromosome (c) Families (d) hsa-mir-2045.441 6.07 0 MI0000284 9q21.11 Yes 43 hsa-mir-203 4.079 2.83 0 MI000028314q32.11 No 113 hsa-mir-211 3.931 2.81 0 MI0000287 15q13.3 Yes 43 Score:T-statistic values. q value: this is the lowest False Discovery Rate atwhich the gene is called significant. It is like the familiar “p-value”,adapted to the analysis of a large number of genes. Active site:indicates if the probe contains the mature form of the microRNA. (d)microRNA Family: related microRNA based on homology in the hairpinflanking reions as reported in The miRBase Sequence Database - Release7.0.

TABLE 8 Differentially expressed MicroRNAs between PETs with differentclinicopathological parameters. Fold Active site microRNA MicroRNA NameScore (a) Change q-value (b) Accession ID Chromosome (c) Families (d)PET with or without liver metastasis (FDR: 0% on the median percentileand 1.8-Fold). Upregulated microRNA hsa-mir-21 2.239446173 1.93 0MI0000077 17q23.2 Yes 61 PET with High (Ki67 >2%) or Low (Ki67 ≦2%)proliferation index (FDR: 0% on the median percentile and 1.8-Fold).Upregulated microRNA hsa-mir-21 2.869445623 1.84 0 MI0000077 17q23.2 Yes61 Non-Functioning PETs with High (Ki67 >2%) or Low (Ki67 ≦2%)proliferation index (FDR: 0% on the median percentile and 2-Fold).Upregulated microRNA hsa-mir-21 2.513043962 2.10 0 MI0000077 17q23.2 Yes61 WDECs with High (Ki67 >2%) or Low (Ki67 ≦2%) proliferation index(FDR: 0% on the median percentile and 2-Fold). Upregulated microRNAhsa-mir-021 1.642156912 2.32 0 MI0000077 17q23.2 Yes 61 Score:T-statistic values. q value: this is the lowest False Discovery Rate atwhich the gene is called significant. It is like the familiar “p-value”,adapted to the analysis of a large number of genes. Active site:indicates if the probe contains the mature form of the microRNA. (d)microRNA Family: related microRNA based on homology in the hairpinflanking reions as reported in The miRBase Sequence Database - Release7.0.

TABLE 9 Putative gene targets of miR-204/211 identified by threeprediction methods Symbol Acc Num UniGene ID Gene Name Ensembl. Gene. IDGene ID Chromosome CDH2 NM_001792 Hs.464829 Cadherin 2, type 1, N-ENSG00000170558 1000 18q11.2 cadherin (neuronal) KHDRBS1 NM_006559Hs.445893 KH domain containing, ENSG00000121774 10657 1p32 RNA binding,signal transduction associated 1 MAPRE2 NM_014268 Hs.532824Microtubule-associated ENSG00000166974 10982 18q12.1 protein, RP/EBfamily, member 2 NCOA7 NM_181782 Hs.171426 Nuclear receptorENSG00000111912 135112 6q22.32 coactivator 7 ATF2 NM_001880 Hs.425104Activating transcription ENSG00000115966 1386 2q32 factor 2 GLIS3NM_152629 Hs.162125 GLIS family zinc finger 3 ENSG00000107249 1697929p24.2 EPHA7 NM_004440 Hs.73962 EPH receptor A7 ENSG00000135333 20456q16.1 C10orf56 NM_153367 Hs.523080 Chromosome 10 open ENSG00000165424219654 10q22.3 reading frame 56 AP3M1 NM_012095 Hs.500104Adaptor-related protein ENSG00000185009 26985 10q22.2 complex 3, mu 1subunit PRO0149 NM_014117 Hs.221497 PRO0149 protein ENSG0000018283129035 16p13.2 NRBF2 NM_030759 Hs.449628 Nuclear receptor bindingENSG00000148572 29982 10q21.3 factor 2 M11S1 NM_005898 Hs.471818Membrane component, ENSG00000135387 4076 11p13 chromosome 11, surfacemarker 1 MYO10 NM_012334 Hs.481720 Myosin X ENSG00000145555 46515p15.1-p14.3 NOVA1 NM_002515 Hs.211225 Neuro-oncological ENSG000001399104857 14q ventral antigen 1 NTRK2 NM_006180 Hs.494312 Neurotrophictyrosine ENSG00000148053 4915 9q22.1 kinase, receptor, type 2 hSynNM_018157 Hs.368253 Brain synembryn ENSG00000111785 55188 12q23.3 HMGA2NM_003483 Hs.505924 High mobility group AT- ENSG00000149948 8091 12q15hook 2 AKAP1 NM_003488 Hs.463506 A kinase (PRKA) anchor ENSG000001210578165 17q21-q23 protein 1 OGT NM_003605 Hs.405410 O-linked N-ENSG00000147162 8473 Xq13 acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide- N- acetylglucosaminyl transferase) CCNT2NM_001241 Hs.292754 Cyclin T2 ENSG00000082258 905 2q21.3

TABLE 10 Putative target genes of miR-204/211 and miR-21 found to bedifferentially expressed Gene Target Fold p. value Putative target genesof miR-204/211 found differentially expressed between NF-PETs andinsulinomas. EST microarray contains 16 out of 20 identified putativetarget genes. UPREGULATED MAPRE2 1.75 0.0070 AP3M1 1.30 0.0330DOWNREGULATED MYO10 0.43 0.0014 AKAP1 0.59 0.0114 Putative target genesof miR-21 found differentially expressed between PET with or withoutliver metastasis. EST microarray contains 11 out of 12 identifiedputative target genes. UPREGULATED NFIB 1.69 0.038 DOWNREGULATED PDCD40.71 0.001 Putative target genes of miR-21 found differentiallyexpressed between PET high (Ki67 > 2) or low (Ki67 ≦ 2). EST microarraycontains 11 out of 12 identified putative target genes. DOWNREGULATEDPDCD4 0.66 0.00001

A Common microRNA Expression Pattern Distinguishes Pancreatic Endocrineand Acinar Tumors from Normal Pancreas.

The vast majority of the differentially expressed microRNAs found inPACC vs. normal tissue were also found in PET vs. normal tissue. Inparticular, 28 of 30 (93%) microRNAs that were overexpressed in PACCwere also found to be upregulated in PET. Similarly, five of seven (71%)underexpressed microRNAs were downregulated in both tumor subtypes. Thisoverlap, together with the fact that only a limited set of microRNAswere differentially expressed between PET and PACC or among PETsubtypes, is suggestive of a pattern of microRNA expression common toacinar and insular-derived tumors.

Among the upregulated microRNAs in PET that are also common to PACC,seven were validated by Northern blot analysis. In particular, miR-103was the best discriminator for all pair-wise comparisons of normalpancreas, acinar cell carcinomas and pancreatic endocrine tumors (FIGS.2A and 2B). The expression of miR-107 paralleled that of its highlyhomologous miR-103, and the significant overexpression of miR-23a,miR-26b, miR-192, and miR-342 in tumors vs. normal was also confirmed(FIGS. 6A-6C).

Among downregulated microRNAs in PET, Northern blot analysis of miR-155showed the lack of detectable expression in both PET and PACC (FIGS. 2Aand 2B). Although miR-155 was not among the top listed downregulatedgenes in PACC (Table 4), its low expression in this tumor type was alsodetected by microarray, as shown in the box-and-whiskers plot of FIG.2A.

A Limited Set of microRNAs Distinguishes Pancreatic Endocrine fromAcinar Tumors.

The direct comparison of PET and PACC showed only 10 upregulatedmicroRNAs (Table 5), all of which were also overexpressed in PET vs.normal tissue. In contrast, no microRNA was found to be specificallyupregulated or downregulated in PACC.

Over-expression of miR-204 is Specific to Insulinomas and Correlateswith Immunohistochemical Expression of Insulin.

The comparison of insulinomas with NF-PET identified only threemicroRNAs that were significantly overexpressed in insulinomas,including miR-204, its homolog miR-211, and miR-203 (Table 7). Notably,the expression of insulin protein, as detected by immunohistochemicalstaining, correlated with miR-204 expression more strongly than withinsulin mRNA expression (FIG. 3A-3C). In fact, logistic regressionanalysis, based on negative or positive ICH staining, showed that theinsulin protein expression was predicted by both insulin mRNA andmiR-204 expression (p<0.001); however, in a multivariate model onlymiR-204 expression retained statistical significance (p<0.001).

As miR-375 was suggested to be specifically expressed in mousepancreatic islets and to function as a negative regulator of insulinexocytosis (Poy, M. N., et al., Nature 432:226-30 (2004)), weinvestigated its expression in normal human tissues and our samples byNorthern blot. Using a panel of several human adult tissues, miR-375 wasonly detected in normal pancreas (FIG. 7A). The expression levels ofmiR-375 were generally higher in tumors vs. normal pancreas, but showedno difference between insulinomas and nonfunctioning tumors (FIGS. 7Band 7C).

Expression of miR-21 is Strongly Associated with the Proliferation Indexand Presence of Liver Metastasis.

The evaluation of expression profiles to identify microRNAsdiscriminating PETs based on either metastatic status or proliferationindex identified only miR-21 as significant (FIGS. 4A-4C and Table 8).This is not surprising, given that these two tumor characteristics areinterconnected. In fact, all metastatic PETs had a proliferationindex >2%, while no tumor with a lower proliferation score wasmetastatic. Furthermore, miR-21 also distinguished between NF-PETs orWDEC with high (Ki-67>2%) and low (Ki-67≦2%) proliferation index.Another interesting observation is that miR-21 was also overexpressed inPACCs versus normal pancreas (Table 4).

Identification of Putative mRNA Targets for Differentially-ExpressedmicroRNAs.

Three different programs (miRanda, TargetScan, PicTar, respectivelyavailable at www.microrna.org/mammalian/index.html;www.genes.mit.edu/targetscan/; and www.pictar.bio.nyu.edu) were used toidentify predicted targets of selected microRNAs, namelymiR-103/miR-107, miR-155, miR-204/miR-211 and miR-21. To increase thestringency of the analysis, we considered only target genes that werefound from all three algorithms (Table 9). Because the same tumorsamples and five normal pancreas analyzed for microRNA expression havealso been evaluated for gene expression profiles with a custom ESTmicroarray (data not shown), we attempted to assess the status ofpredicted mRNA targets in PET and normal tissue, as well as among PETwith different clinicopathological characteristics. A two-sample-t-testanalysis identified several putative target genes that were eitherdownregulated or upregulated, namely 28 upregulated and 7 downregulatedgenes for miR-103/107, 2 upregulated and 2 down-regulated genes foreither miR-155 or miR-204/211, and 1 upregulated and 1 downregulatedgene for miR-21 (Table 10). Notably, the mRNA expression of PDCD4 gene,a putative target of miR-21, was found to be downregulated in livermetastatic PET, as well as in tumors with high proliferation index,showing an inverse correlation with the expression of miR-21 (FIG. 5).

Discussion

The results of the survey of microRNA expression profiles in normalpancreas, pancreatic endocrine tumors and acinar carcinomas may besummarized as follows:

a common microRNA expression profile distinguishes both endocrine andacinar tumors from normal pancreas;

the expression of miR-103 and miR-107 associated with lack of expressionof miR-155 discriminates tumors from normal;

a limited set of microRNAs is specific to endocrine tumors and ispossibly associated with the endocrine differentiation or tumorigenesis;

miR-204 expression occurs primarily in insulinomas and correlates withimmunohistochemical expression of insulin; and

expression of miR-21 is strongly associated with proliferation index andliver metastasis.

Unsupervised hierarchical clustering of the expression profiles showedthat both tumor types were separated from normal pancreas. AlthoughPACCs fell into a unique cluster, this was part of the wider clusterincluding all PETs. While we identified many more differentiallyexpressed microRNAs in PET versus normal than between acinar carcinomasversus normal, the vast majority of differentially expressed microRNAsin PACC were similarly altered in PET. It is worth noting that bulkpancreas is largely formed by acini and therefore represents the idealnormal counterpart for the analysis of acinar cell carcinomas, whilepancreatic islet cells would represent the normal counterpart forpancreatic endocrine tumors. Unfortunately, we had no preparations ofthese cells available. Nonetheless, the finding of a largely concordantpattern of differentially expressed microRNAs between acinar and insulartumors, including 28 upregulated and 5 downregulated genes, suggeststhat this set common to both tumor types might be related to pancreaticneoplastic transformation. Providing additional support for thisassertion, several microRNAs differentially expressed in both tumortypes have been found to be differentially expressed in breast, colonand B-cell leukemia (Caldas, C., et al., Nat. Med. 11:712-14 (2005);Croce, C. M., and G. A. Calin, Cell 122:6-7 (2005); Iorio, M. V., etal., Cancer Res. 65:7065-70 (2005)). In addition, at least twenty of thedifferentially-expressed microRNAs in our tumors have been identified ashaving either growth related or apoptotic effects in the lung A549 orcervical HeLa carcinoma cell lines (Cheng, A. M., et al., Nucleic AcidsRes. 33:1290-97 (2005)).

Furthermore, we observed, in both PACC and PET, the coordinateoverexpression of miR-17, miR-20 and miR-92-1, which are contained in apolycistronic cluster. This miR-17-92 cluster has been described to actas an oncogene in association with c-MYC gene (He, L., et al., Nature435:828-33 (2005)). Notably, overexpression of c-MYC has been reportedin pancreatic endocrine tumors and also in hyperplastic islets,suggesting its involvement in the early phases of insular tumorigenesis(Pavelic, K., et al., Anticancer Res. 16:1707-17 (1996)). In addition,induction of MYC in islet or acinar cells of mouse in in vitro or invivo models produces endocrine tumors (Katic, M., et al., Carcinogenesis20:1521-27 (1999); Lewis, B. C., et al., Genes Dev. 17:3127-38 (2003))or mixed acinar/ductal adenocarcinomas (Sandgren, E. P., et al., Proc.Natl. Acad. Sci. USA 88:93-97 (1991), respectively, while suppression ofMYC-induced apoptosis leads to islet cells carcinoma (Pelengaris, S., etal., Cell 109:321-34 (2002)).

The expression of the two highly homologous miR-103 and miR-107microRNAs together with the lack of expression of miR-155 wasdistinctive of tumors vs. normal pancreatic samples. Interestingly,miR-103/107 have been found to be overexpressed in several tumor types(entitled “Micro-RNA-Based Methods and Compositions for the Diagnosisand Treatment of Solid Cancers”, by Stefano Volinia, George A. Calin andCarlo M. Croce; US Pub. No. 2008/0306006; filed on same date as thesubject application; the teachings of which are incorporated herein byreference in their entirety). The finding that miR-155 was expressed innormal pancreas but was underexpressed or not expressed in both PET andPACC is rather interesting considering that overexpression of miR-155has been observed in lymphomas (Caldas, C., et al., Nat. Med. 11:712-14(2005); Croce, C. M., and G. A. Calin, Cell 122:6-7 (2005)) and breastcancer (Iorio, M. V., et al., Cancer Res. 65:7065-70 (2005)), a findingthat has led to speculation that miR-155 may be an oncogenic microRNA(Croce, C. M., and G. A. Calin, Cell 122:6-7 (2005)). This may not beunexpected, as microRNAs expressed in adults are tissue-specific (Babak,T., et al., RNA 10:1813-19 (2004)) and the consequences of microRNAmisexpression is highly dependent on the cell-specific expressionpattern of mRNAs that are microRNA regulated (Cheng, A. M., et al.,Nucleic Acids Res. 33:1290-97 (2005)).

Ten microRNAs were peculiarly overexpressed in PET and differentiatedthis tumor from both PACC and normal pancreas. These included miR-99a,miR-99b, miR-100, miR-125a, miR-125b-1, miR-125b-2, miR-129-2, miR-130a,miR-132, and miR-342. These microRNAs may be characteristic of eitherendocrine differentiation or endocrine tumorigenesis. On the other hand,no microRNA was found to be specifically upregulated or downregulated inPACC, although the limited number of PACC samples may have affected thepower of the analysis.

Although the microRNA profiles were almost indistinguishable betweeninsulinomas and nonfunctioning endocrine tumors, the overexpression ofthe two closely related microRNAs, namely miR-204 and miR-211, waslargely restricted to insulinomas. Of great interest, miR-204 expressioncorrelated with the immunohistochemical expression of insulin. In thisrespect, miR-375 has been recently reported to be specifically expressedin mouse pancreatic islets and to function as a negative regulator ofinsulin exocytosis (Poy, M. N., et al., Nature 432:226-30 (2004)). Ourdata showed that this microRNA is expressed in human normal pancreas, aswell as in acinar cell and endocrine tumors. However, no difference wasfound in its expression level between insulinomas and nonfunctioningendocrine tumors.

We also determined if microRNA expression was correlated with theclinical characteristics of PETs. Our results showed that miR-21overexpression is associated with both enhanced Ki-67 proliferationindex and liver metastasis. miR-21 overexpression has been observed inseveral cancers, including glioblastoma, breast, lung and colon cancers(Caldas, C., et al., Nat. Med. 11:712-14 (2005); Croce, C. M., and G. A.Calin, Cell 122:6-7 (2005)). A cancer-related function of miR-21 is alsosupported by knockdown experiments in glioblastoma cell lines showingthat this microRNA has an anti-apoptotic function (Chan, J. A., et al.,Cancer Res. 65:6029-33 (2005)). In this respect, the programmed celldeath 4 (PDCD4) gene, putatively targeted by miR-21, was found to besignificantly downregulated in metastatic and high proliferative PETsamples, and showed an inverse correlation with the expression ofmiR-21. This gene has been reported to act as a tumor suppressor throughactivation of p21 waf1 and inhibition of transcription factor complexAP-1; the latter controls genes that have been implied in cellularinvasion and metastatic progression (Jansen, A. P., et al., Mol. Cancer.Ther. 3:103-10 (2004)). Furthermore, PDCD4 expression is lost inprogressed carcinomas of lung, breast, colon and prostate cancer (Goke,R., et al., Am. J. Physiol. Cell Physiol. 287:C1541-46 (2004)), andnotably, a tumor suppressor role for PDCD4 has been also reported in amodel of neuroendocrine tumor cells (Goke, R., et al., Ann. N.Y. Acad.Sci. 1014:220-21 (2004)).

Differentially-expressed microRNAs in PETs showed a nonrandomdistribution among chromosomal arms and most of the microRNAs located atchromosomal arms 5q, 7q, 13q and 19p were overexpressed. This findingmay be due to either the frequent association of microRNAs inpolycistronic clusters (Baskerville, S, and D. P. Bartel, RNA 11:241-47(2005); Altuvia, Y., et al., Nucleic Acids Res. 33:2697-2706 (2005)) orthe amplification of the chromosomal arms containing these microRNAs.Our analysis suggests that both phenomena can be involved in PET. Infact, the correlation coefficients measured between pairs of microRNAswithin clusters differed significantly from those between pairs ofmicroRNAs outside the clusters. These data confirm in PET the generalobservation that grouped microRNA genes show coordinate expression(Baskerville, S, and D. P. Bartel, RNA 11:241-47 (2005); Altuvia, Y., etal., Nucleic Acids Res. 33:2697-2706 (2005)).

MicroRNAs exert their biological effects by targeting specific mRNAs fordegradation or translational inhibition. In order to get insights intothe biological implications of the most interesting microRNAs showingaltered expression in pancreatic tumors, e.g., miR-103/miR-107, miR-155,miR-204/miR-211 and miR-21, we searched predicted targets that were incommon among those identified by three different algorithms (seeRESULTS). Then, to evaluate if there was a correlation between theexpression of microRNAs and that of their predicted targets, we tookadvantage of the EST expression profiles of the same tumor and normalsamples. Among the selected targets that were contained in our ESTmicroarray, we found several upregulated and downregulated genes.Interestingly, the predicted target genes of miR-103/107 wereoverexpressed more frequently than expected. This finding parallels thatof Babak et al., who reported a low correlation between microRNAexpression and their predicted mRNA targets in a set of 17 differentmouse tissues (Babak, T., et al., RNA 10:1813-19 (2004)). This supportsthe currently favored model that most microRNAs act more likely throughtranslational inhibition without mRNA degradation (Bartel, D. P., Cell116:281-97 (2004)).

In conclusion, the results described herein suggest that alteration inmicroRNA expression is related to endocrine and acinar neoplastictransformation and progression of malignancy.

The relevant teachings of all publications cited herein that have notexplicitly been incorporated by reference, are incorporated herein byreference in their entirety. In addition, the nucleotide sequences(e.g., microRNA nucleotide sequences) identified herein by reference tospecific Accession Number are also incorporated herein by reference intheir entirety. While this invention has been particularly shown anddescribed with references to preferred embodiments thereof, it will beunderstood by those skilled in the art that various changes in form anddetails may be made therein without departing from the scope of theinvention encompassed by the appended claims.

What is claimed is:
 1. A method of determining whether a subject havinga pancreatic cancer has, or is at risk for developing, a livermetastasis, the method comprising: extracting from the subject a testsample comprising a miR-21 gene product; measuring the level of the geneproduct in the test sample; determining whether the subject has, or isat risk for developing, pancreatic cancer by comparing the level of thegene product in the test sample to the level of a corresponding miR geneproduct in a control sample; and, correlating an increase in the levelof the miR-103 gene product in the test sample, relative to the level ofthe corresponding miR gene product in the control sample, as beingindicative of a diagnosis of the subject either having, or being at riskfor developing, a liver metastasis.
 2. The method according to claim 1,wherein the measuring the level of the miR-21 gene product in the testsample is carried out by analyzing the test sample with a RT-PCR, amicroarray and/or a Northern blot electrophoretic device.
 3. The methodaccording to claim 1, further comprising: confirming diagnosis bysearching, or periodically monitoring, for symptoms which are associatedwith pancreatic cancer and exhibited by the subject when there is anincrease in the level of the miR-21 gene product in the test sample,relative to the level of the corresponding miR gene product in thecontrol sample.
 4. The method according to claim 1, wherein thepancreatic cancer is a pancreatic endocrine tumor (PET).
 5. The methodaccording to claim 4, wherein the pancreatic cancer is at least onepancreatic endocrine tumor (PET) selected from the group consisting ofgastrinoma, insulinoma, somatostatinoma, VIPoma, and glucagonoma.
 6. Themethod according to claim 1, further comprising: confirming diagnosis bysearching, or periodically monitoring, for symptoms of hormonehypersecretion associated with PET and exhibited by the subject whenthere is an increase in the level of the miR-21 gene product in the testsample, relative to the level of the corresponding miR gene product inthe control sample.
 7. The method according to claim 6, wherein thehormone is at least one hypersecreted hormone selected from the groupconsisting of gastrin, insulin, somatostatin, vasoactive intestinalpolypeptide (VIP), and glucagon.
 8. The method according to claim 1,further comprising: communicating to the subject the diagnosis ofhaving, or being at risk for developing, liver metastasis of pancreaticcancer when there is an increase in the level of the miR-21 gene productin the test sample, relative to the level of the corresponding miR geneproduct in the control sample.
 9. A method of determining whether asubject having a pancreatic cancer has, or is at risk for developing, aliver metastasis, the method comprising: isolating RNA from a testsample extracted from the subject; reverse transcribing the RNA isolatedfrom the test sample to provide at least one targetoligodeoxynucleotide; hybridizing the at least one targetoligodeoxynucleotide to a microarray comprising at least one miR-21miRNA-specific probe oligonucleotide to provide a hybridization profilefor the test sample; determining whether the subject has, or is at riskfor developing, a liver metastasis of pancreatic cancer by comparing thesignal of the miR-21 miRNA in the hybridization profile for the testsample to the signal of a corresponding miRNA in the hybridizationprofile for a control sample; and, correlating a up-regulated signal ofthe miR-103 miRNA in the test sample, relative to the signal of thecorresponding miRNA in the control sample, as being indicative of adiagnosis of the subject either having, or being at risk for developing,a liver metastasis of pancreatic cancer.
 10. The method according toclaim 9, further comprising: confirming diagnosis by searching, orperiodically monitoring, for symptoms which are associated withpancreatic cancer and exhibited by the subject when there is anupregulated signal of the miR-21 miRNA in the test sample, relative tothe signal of the corresponding miRNA in the control sample.
 11. Themethod according to claim 9, wherein the pancreatic cancer is apancreatic endocrine tumor (PET).
 12. The method according to claim 9,wherein the pancreatic cancer is at least one pancreatic endocrine tumor(PET) selected from the group consisting of gastrinoma, insulinoma,somatostatinoma, VIPoma, and glucagonoma.
 13. The method according toclaim 12, further comprising: confirming diagnosis by searching, orperiodically monitoring, for symptoms of hormone hypersecretionassociated with PET and exhibited by the subject when there is anupregulated signal of the miR-21 miRNA in the test sample, relative tothe signal of the corresponding miRNA in the control sample.
 14. Themethod according to claim 13, wherein the hormone is at least onehypersecreted hormone selected from the group consisting of gastrin,insulin, somatostatin, vasoactive intestinal polypeptide (VIP), andglucagon.